CN103025890A

CN103025890A - Circulating biomarkers for disease

Info

Publication number: CN103025890A
Application number: CN2011800275418A
Authority: CN
Inventors: C·库斯利驰; G·波斯特; M·克拉斯; D·斯佩兹勒; T·波洛斯基
Original assignee: Caris Life Sciences Luxembourg Holdings SARL
Current assignee: Caris Life Sciences Luxembourg Holdings SARL
Priority date: 2010-04-06
Filing date: 2011-04-06
Publication date: 2013-04-03
Anticipated expiration: 2031-04-06

Abstract

Biomarkers can be assessed for diagnostic, therapy-related or prognostic methods to identify phenotypes, such as a condition or disease, or the stage or progression of a disease. Circulating biomarkers from a bodily fluid can be used in profiling of physiological states or determining phenotypes. These include nucleic acids, protein, and circulating structures such as vesicles. Biomarkers can be used for theranostic purposes to select candidate treatment regimens for diseases, conditions, disease stages, and stages of a condition, and can also be used to determine treatment efficacy. The biomarkers can be circulating biomarkers, including vesicles and microRNA.

Description

The circulating biological mark of disease

Cross reference

The application requires the No.61/321 submitted on April 6th, 2010,392, the No.61/332 that on May 6th, 2010 submits to, 174, the No.61/348 that on May 25th, 2010 submits to, 214, the No.61/348 that on May 26th, 2010 submits to, 685, the No.61/354 that on June 11st, 2010 submits to, 125, the No.61/355 that on June 16th, 2010 submits to, 387, the No.61/362 that on July 8th, 2010 submits to, 674, the No.61/413 that on November 12nd, 2010 submits to, 377, the No.61/322 that on April 9th, 2010 submits to, 690, the No.61/334 that on May 13rd, 2010 submits to, 547, the No.61/370 that on August 2nd, 2010 submits to, 088, the No.61/391 that on October 8th, 2010 submits to, 504, the No.61/393 that on October 15th, 2010 submits to, 823, the No.61/416 that on November 23rd, 2010 submits to, 560, the No.61/321 that on April 6th, 2010 submits to, 407, the No.61/356 that on June 21st, 2010 submits to, 974, the No.61/379 that on September 2nd, 2010 submits to, 670, the No.61/381 that on September 9th, 2010 submits to, 305, the No.61/383 that on September 15th, 2010 submits to, the No.61/411 that on November 9th, 305 and 2010 submits to, the rights and interests of 890 U.S. Provisional Patent Application.

Background of invention

For such as the situation of cancer and the biomarker of disease, comprising biomolecules, such as protein, peptide, lipid, RNA, DNA and their variant and modification.

The evaluation of particular organisms mark (for example DNA, RNA and protein) can be provided for the biological marking of diagnosis, prognosis or treatment diagnosis (theranosis) situation or disease.Biomarker can detect in body fluid, comprises Circulating DNA, RNA, protein and vesica.The circulating biological mark comprises protein (such as PSA and CA125) and nucleic acid (such as SEPT9DNA and PCA3 messenger RNA(mRNA) (mRNA)).The circulating biological mark also comprises circulating vesica.Vesica is the structure that the film that comes off from cell is sealed, and is found in multiple body fluid, comprises blood, blood plasma, serum, milk, ascites, bronchoalveolar lavage fluid and urine.Vesica can be used as protein, RNA, DNA, virus and the transporting carrier of Protein virus and participates in the communication between cell.MicroRNA is the short rna that the regulation and control messenger RNA(mRNA) is transcribed and degraded.MicroRNA is found and has been observed it as the component in the vesica come off from tumour cell in body fluid.Analysis to the circulating biological mark with disease-related (comprising vesica and/or microRNA) can contribute to detect disease or its severity, determines the susceptibility of disease and treated decision-making.

The vesica existed in biological sample provides the source of biomarker, and for example, described mark is present in vesica interior (vesica useful load) or is present on the surface of vesica.The feature of vesica (for example definite, the useful load of size, surface antigen, cell source) also can provide the result output of diagnosis, prognosis or treatment diagnosis.Still the demand that exists the biomarker to can be used for the test-and-treat disease to be identified.The feature of the microRNA relevant to vesica and other biomarker and vesica can provide diagnosis, prognosis or treatment diagnosis.

The invention provides for characterize the method and system of phenotype as the biomarker of the indication of disease or progression of disease by detection.Described biomarker can be the circulating biological mark, comprises vesica and microRNA.

Summary of the invention

Herein disclosed is for characterize the method and composition of phenotype by analyzing vesica (such as the vesica be present in from the biological sample of experimenter's cell).Characterize the determining of prognosis, disease stage or situation stage, pharmaceutical efficacy, physiological situation that experimenter or individual phenotype can include but not limited to diagnosis, disease or the situation of disease or situation, organ danger is tired or organ rejection, disease or progress, with the relevant cognation for the treatment of of disease or situation or specifically physiology or biological aspect.

In one aspect, the invention provides the method that characterizes the prostate gland imbalance, comprise existence or the level determined from one or more biomarkers in experimenter's biological sample, the biological marking of the existence that evaluation comprises described one or more biomarkers or level, and the described biological marking and reference are compared, characterize therefrom described prostate gland imbalance.Available biomarker is listed in this paper table 5 or table 9-11.The step that the described biological marking and described reference are compared can determine whether any biomarker in described one or more biomarkers changes to some extent with respect to described reference, and determining of prognosis, diagnosis or treatment diagnosis to described prostate gland imbalance is provided thus.

In one embodiment, described prostate gland imbalance comprises that BPH and described one or more biomarkers are selected from BCMA, CEACAM-1, HVEM, IL-1R4, IL-10Rb, Trappin-2, p53, hsa-miR-329, hsa-miR-30a, hsa-miR-335, hsa-miR-152, hsa-miR-151-5p, hsa-miR-200a, hsa-miR-145, hsa-miR-29a, hsa-miR-106b, hsa-miR-595, hsa-miR-142-5p, hsa-miR-99a, hsa-miR-20b, hsa-miR-373, hsa-miR-502-5p, hsa-miR-29b, hsa-miR-142-3p, hsa-miR-663, hsa-miR-423-5p, hsa-miR-15a, hsa-miR-888, hsa-miR-361-3p, hsa-miR-365, hsa-miR-10b, hsa-miR-199a-3p, hsa-miR-181a, hsa-miR-19a, hsa-miR-125b, hsa-miR-760, hsa-miR-7a, hsa-miR-671-5p, hsa-miR-7c, hsa-miR-1979, hsa-miR-103 and combination thereof.

In another embodiment, described prostate gland imbalance comprises and comprises that prostate cancer and described one or more biomarkers are selected from CD9, PSMA, PCSA, CD63, CD81, B7H3, IL6, OPG-13, IL6R, PA2G4, EZH2, RUNX2, SERPINB3, EpCam, hsa-let-7b, hsa-miR-107, hsa-miR-1205, hsa-miR-1270, hsa-miR-130b, hsa-miR-141, hsa-miR-143, hsa-miR-148b ^*, hsa-miR-150, hsa-miR-154 ^*, hsa-miR-181a ^*, hsa-miR-181a-2 ^*, hsa-miR-18a ^*, hsa-miR-19b-1 ^*, hsa-miR-204, hsa-miR-2110, hsa-miR-215, hsa-miR-217, hsa-miR-219-2-3p, hsa-miR-23b ^*, hsa-miR-299-5p, hsa-miR-301a, hsa-miR-301a, hsa-miR-326, hsa-miR-331-3p, hsa-miR-365 ^*, hsa-miR-373 ^*, hsa-miR-424, hsa-miR-424 ^*, hsa-miR-432, hsa-miR-450a, hsa-miR-451, hsa-miR-484, hsa-miR-497, hsa-miR-517 ^*, hsa-miR-517a, hsa-miR-518f, hsa-miR-574-3p, hsa-miR-595, hsa-miR-617, hsa-miR-625 ^*, hsa-miR-628-5p, hsa-miR-629, hsa-miR-634, hsa-miR-769-5p, hsa-miR-93, hsa-miR-96 and combination thereof.The imbalance of described prostate gland comprises and can be prostate cancer and described one or more biomarkers can be selected from A33, a33n15, AFP, ALA, ALIX, ALP, annexin V, APC, ASCA, ASPH (246-260), ASPH (666-680), ASPH (A-10), ASPH (D01P), ASPH (D03), ASPH (G-20), ASPH (H-300), AURKA, AURKB, B7H3, B7H4, BCA-225, BCNP1, BDNF, BRCA, CA125 (MUC16), CA-19-9, C-Bir, CD1.1, CD10, CD174 (Lewis y), CD24, CD44, CD46, CD59 (MEM-43), CD63, CD66e CEA, CD73, CD81, CD9, CDA, CDAC1 1a2, CEA, C-Erb2, C-erbB2, CRMP-2, CRP, CXCL12, CYFRA21-1, DLL4, DR3, EGFR, Epcam, EphA2, EphA2 (H-77), ER, ErbB4, EZH2, FASL, FRT, FRT c.f23, GDF15, GPCR, GPR30, Gro-α, HAP, HBD 1, HBD2, HER 3 (ErbB3), HSP, HSP70, hVEGFR2, iC3b, IL 6Unc, IL-1B, IL6 Unc, IL6R, IL8, IL-8, INSIG-2, KLK2, L1CAM, LAMN, LDH, MACC-1, MAPK4, MART-1, MCP-1, M-CSF, MFG-E8, MIC1, MIF, MIS RII, MMG, MMP26, MMP7, MMP9, MS4A1, MUC1, MUC1 seq1, MUC1 seq11A, MUC17, MUC2, Ncam, NGAL, NPGP/NPFF2, OPG, OPN, p53, p53, PA2G4, PBP, PCSA, PDGFRB, PGP9.5, PIM1, PR (B), PRL, PSA, PSMA, PSME3, PTEN, R5-CD9 Tube 1, Reg IV, RUNX2, SCRN1, seprase, SERPINB3, SPARC, SPB, SPDEF, SRVN, STAT 3, STEAP1, TF (FL-295), TFF3, TGM2, TIMP-1, TIMP1, TIMP2, TMEM211, TMPRSS2, TNF-α, Trail-R2, Trail-R4, TrKB, TROP2, Tsg 101, TWEAK, UNC93A, VEGF A, YPSMA-1 and combination thereof.

Described sign can comprise the biological marking of prostate cancer in the described sample of detection.Characterize prostate cancer and can comprise by described characterizing prostate cancer being metastatic or invasive.In these cases, described one or more biomarkers can be selected from hsa-miR-100, hsa-miR-1236, hsa-miR-1296, hsa-miR-141, hsa-miR-146b-5p, hsa-miR-17 ^*, hsa-miR-181a, hsa-miR-200b, hsa-miR-20a ^*, hsa-miR-23a ^*, hsa-miR-331-3p, hsa-miR-375, hsa-miR-452, hsa-miR-572, hsa-miR-574-3p, hsa-miR-577, hsa-miR-582-3p, hsa-miR-937, miR-10a, miR-134, miR-141, miR-200b, miR-30a, miR-32, miR-375, miR-495, miR-564, miR-570, miR-574-3p, miR-885-3p and combination thereof.

Described sign also can comprise determines that described experimenter processes and whether just reacts for treatment, or described experimenter processes and may respond or reactionless for treatment.In one embodiment, the table 8-10 that is selected from this paper is processed in described treatment.For example, described treatment pack processing is containing radical prostatectomy and/or radiotherapy.The nursing standard that can be selected from prostate cancer is processed in described treatment, it includes but not limited to observe and waits for, the surgery pelvic lymphadenectomy, radical prostatectomy, transurethral prostatectomy (TURP), testectomy, radiotherapy, external radiation exposure radiotherapy (EBRT), iodine I 125, palladium, iridium, hormonotherapy, the bright dried meat Li Te of luteinising hormone-releasing hormo agonist (leuprolide), goserelin, buserelin (buserelin), antiandrogen, flutamide, bicalutamide, Magace, Nilutamide, KETOKONAZOL, aminoglutethimide, gonadotropin releasing hormone (GnRH), oestrogenic hormon, psychrotherapy, chemotherapy, biotherapy, one or more in ultrasonic and proton beam radiation.Can comprise one or more of hsa-miR-1974, hsa-miR-27b, hsa-miR-103, hsa-miR-146a, hsa-miR-22, hsa-miR-382, hsa-miR-23a, hsa-miR-376c, hsa-miR-335, hsa-miR-142-5p, hsa-miR-221, hsa-miR-142-3p, hsa-miR-151-3p, hsa-miR-21 and hsa-miR-16 for monitoring to the biological marking of reaction for the treatment of.

In some embodiments of the present invention, described one or more biomarkers comprise 5T4, ACTG1, ADAM10, ADAM15, ALDOA, ANXA2, ANXA6, APOA1, ATP1A1, BASP1, Clorf58, C20orfl14, C8B, CAPZA1, CAV1, CD151, CD2AP, CD59, CD9, CD9, CFL1, CFP, CHMP4B, CLTC, COTL1, CTNND1, CTSB, CTSZ, CYCS, DPP4, EEF1A1, EHD1, ENO1, F11R, F2, F5, FAM125A, FNBP1L, FOLH1, GAPDH, GLB1, GPX3, HIST1H1C, HIST1H2AB, HSP90AB1, HSPA1B, HSPA8, IGSF8, ITGB1, ITIH3, JUP, LDHA, LDHB, LUM, LYZ, MFGE8, MGAM, MMP9, MYH2, MYL6B, NME1, NME2, PABPC1, PABPC4, PACSIN2, PCBP2, PDCD6IP, PRDX2, PSA, PSMA, PSMA1, PSMA2, PSMA4, PSMA6, PSMA7, PSMB1, PSMB2, PSMB3, PSMB4, PSMB5, PSMB6, PSMB8, PTGFRN, RPS27A, SDCBP, SERINC5, SH3GL1, SLC3A2, SMPDL3B, SNX9, TACSTD1, TCN2, THBS1, TPI1, TSG101, TUBB, VDAC2, VPS37B, YWHAG, one or more in YWHAQ and YWHAZ.Described one or more biomarkers also can comprise one or more in CD9, CD63, CD81, PSMA, PCSA, B7H3 and EpCam.Each of these biomarkers all can be combined and be assessed with the vesica film.

In yet another aspect, the invention provides the method that characterizes benign prostatic hyperplasia (BPH), comprise existence or the level determined from one or more biomarkers listed in this paper table 5 in experimenter's biological sample, the biological marking of the existence that evaluation comprises described one or more biomarkers or level, and the described biological marking and reference are compared.The present invention also provides the method that characterizes prostate cancer, comprise existence or the level determined from one or more biomarkers listed in this paper table 5 in experimenter's biological sample, the biological marking of the existence that evaluation comprises described one or more biomarkers or level, and the described biological marking and reference are compared.Again in addition, the invention provides the invasive method that characterizes prostate cancer, comprise existence or the level determined from one or more biomarkers listed in this paper table 5 in experimenter's biological sample, the biological marking of the existence that evaluation comprises described one or more biomarkers or level, and the described biological marking and reference are compared.

In the embodiment of described method, described biological sample comprises body fluid.Described body fluid can comprise peripheral blood, serum, blood plasma, ascites, urine, cerebrospinal fluid (CSF), phlegm, saliva, marrow, synovia, aqueous humor, amniotic fluid, earwax, milk, bronchoalveolar lavage fluid, seminal fluid, prostatic fluid, examine amber liquid (cowper ' s fluid) or the liquid of ejaculating in advance, the women penetrates liquid, sweat, movement, hair, tear, capsule liquid, hydrothorax and ascites fluid, pericardial fluid, lymph liquid, the food gruel, chyle, bile, interstitial fluid, menses, purulence, sebum, vomitus, vaginal secretions, mucous membrane secretory product, just rare, pancreatic juice, nasal lavage fluid, the segmental bronchus aspirated liquid, blastochyle or Cord blood.Described biological sample can be, for example urine, blood or blood derivatives.Blood derivatives includes but not limited to blood plasma and serum.

In some embodiments, described biological sample comprises one or more microcapsule bubbles.In the described biological marking, one or more included biomarkers can be associated with described one or more microcapsule bubbles.When the described biological marking comprises multiple markers, some marks can associated vesica, and other mark is not associated.Perhaps, the whole marks in the described biological marking can be associated with described one or more microcapsule bubbles.

Described one or more microcapsule bubbles can have the diameter of 20nm to 800nm, for example, and 20nm to 200nm, 20nm to 100nm or 100nm to 500nm.

Can carry out that size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanometer film ultrafiltration, immunosorption are caught to described one or more microcapsule bubbles, affinity purification, affinity capture, immunoassay, microfluidic separation, flow cytometry or their combination.These methods can be used for intactly or partly other composition in described one or more microcapsule bubbles and described sample are separated.

In some embodiments, described one or more microcapsule bubbles are contacted with one or more wedding agents.Described one or more wedding agents can comprise nucleic acid, DNA molecular, RNA molecule, antibody, antibody fragment, fit, class peptide, zDNA, peptide nucleic acid(PNA) (PNA), lock nucleic acid (LNA), lectin, peptide, arborescence, membrane protein labelling thing, chemical compound or its combination.Described one or more wedding agents can be used for catching and/or detect described one or more microcapsule bubbles.For example, described one or more wedding agents can be incorporated into one or more surface antigens on described one or more microcapsule bubbles.Described one or more wedding agents can be used for catching described one or more microcapsule bubbles, and for example, wherein said wedding agent is incorporated into substrate.Described one or more wedding agents also can be used for detecting described one or more microcapsule bubbles, and for example, wherein said wedding agent mark is maybe can also be incorporated into mark part.

One or more surface antigens by described wedding agent identification can be one or more protein, for example, and the surface protein on described microcapsule bubble.Described one or more protein can comprise one or more in CD9, CD63, CD81, PSMA, PCSA, B7H3 and EpCam.Described one or more protein can comprise one or more in the listed protein of four transmembrane proteins, CD9, CD63, CD81, CD63, CD9, CD81, CD82, CD37, CD53, Rab-5b, annexin V, MFG-E8 or table 3.

Except surface antigen, described one or more biomarkers can comprise the useful load in described one or more microcapsule bubbles.For example, surface antigen can be used for catching described one or more microcapsule bubbles, estimates subsequently the useful load in one or more microcapsule bubbles of catching.

Described microcapsule bubble useful load can comprise one or more nucleic acid, peptide, protein, lipid, antigen, carbohydrate and/or proteoglycan.Described nucleic acid can comprise one or more DNA, mRNA, microRNA, snoRNA, snRNA, rRNA, tRNA, siRNA, hnRNA or shRNA.In one embodiment, described nucleic acid comprises one or more microRNAs, and it is selected from hsa-miR-200b, hsa-miR-375, hsa-miR-141, hsa-miR-331-3p, hsa-miR-181a, hsa-miR-574-3p and combination thereof.Described nucleic acid also can comprise one or more microRNAs that are selected from this paper table 27-41.

In yet another aspect, the invention provides the method that characterizes colorectal carcinoma, described method comprises, determine existence or the level of one or more biomarkers in the biological sample from the experimenter, the existence that evaluation comprises one or more biomarkers in described biological sample or the biological marking of level, and the described biological marking and reference are compared, characterized therefrom described colorectal carcinoma.Available biomarker is listed in this paper table 6 and table 9-11.Whether the step that the described biological marking is compared with described reference can comprise the relative described reference of any biomarker of determining in described one or more biomarkers and change to some extent, and prognosis, the diagnosis to described colorectal carcinoma is provided thus or has treated determining of diagnosis.

In one embodiment, described one or more biomarkers are selected from miR-92, miR-21, miR-9, miR-491, hsa-miR-376c, hsa-miR-215, hsa-miR-652, hsa-miR-582-5p, hsa-miR-324-5p, hsa-miR-1296, hsa-miR-28-5p, hsa-miR-190, hsa-miR-590-5p, hsa-miR-202, hsa-miR-195 and combination thereof.In another embodiment, described one or more biomarkers comprise one or more in Muc1, GPCR 110, TMEM211 and CD24.In another embodiment, described one or more biomarkers comprise A33, AFP, ALIX, ALX4, ANCA, APC, ASCA, AURKA, AURKB, B7H3, BANK1, BCNP1, BDNF, CA-19-9, CCSA-2, CCSA-3& 4, CD10, CD24, CD44, CD63, CD66 CEA, CD66e CEA, CD81, CD9, CDA, C-Erb2, CRMP-2, CRP, CRTN, CXCL12, CYFRA21-1, DcR3, DLL4, DR3, EGFR, Epcam, EphA2, FASL, FRT, GAL3, GDF15, GPCR (GPR110), GPR30, GRO-1, HBD 1, HBD2, HNP1-3, IL-1B, IL8, IMP3, L1CAM, LAMN, MACC-1, MGC20553, MCP-1, M-CSF, MIC1, MIF, MMP7, MMP9, MS4A1, MUC1, MUC17, MUC2, Ncam, NGAL, NNMT, OPN, p53, PCSA, PDGFRB, PRL, PSMA, PSME3, Reg IV, SCRN1, Sept-9, SPARC, SPON2, SPR, SRVN, TFF3, TGM2, TIMP-1, TMEM211, TNF-α, TPA, TPS, Trail-R2, Trail-R4, TrKB, TROP2, Tsg 101, TWEAK, one or more in UNC93A and VEGFA.Described one or more biomarkers can comprise one or more in CD9, EGFR, NGAL, CD81, STEAP, CD24, A33, CD66E, EPHA2, ferritin, GPR30, GPR110, MMP9, OPN, p53, TMEM211, TROP2, TGM2, TIMP, EGFR, DR3, UNC93A, MUC17, EpCAM, MUC1, MUC2, TSG101, CD63, B7H3, CD24 and TETS.

Described sign can be included in described sample and detect the biological marking of colorectal carcinoma.Described sign can comprise described colorectal carcinoma is accredited as metastatic or invasive.

Whether described sign also can comprise measures described experimenter and processes and respond for treatment, or whether described experimenter processes and likely respond or reactionless for treatment.Described treatment is processed can be selected from this paper table 8-10.It can be the standard of care of described colorectal carcinoma that described treatment is processed, it includes but not limited to one or more main operative therapys, local excision, the removal of the excision of primary lesion and anastomosis and peripheral lymphoglandula, adjuvant therapy, Fluracil (5-FU), capecitabine (capecitabine), folinic acid, oxaliplatin (oxaliplatin), Tarceva (erlotinib), irinotecan (irinotecan), acetylsalicylic acid, ametycin, Sutent (suntinib), Cetuximab (cetuximab), rhuMAb-VEGF (bevacizumab), filgrastim (pegfilgrastim), Victibix, draw wooden Xidan to resist (ramucirumab), celecoxib, combination treatment, the FOLFOX4 therapy, the FOLFOX6 therapy, the FOLFIRI therapy, the FUFOX therapy, the FUOX therapy, the IFL therapy, the XELOX therapy, 5-FU and LEVAMISOLE HCL therapy, German AIO therapy, the CAPOX therapy, Douillard therapy and radiotherapy.

In one embodiment, described one or more biomarkers comprise A26C1A, A26C1B, A2M, ACAA2, ACE, ACOT7, ACP1, ACTA1, ACTA2, ACTB, ACTBL2, ACTBL3, ACTC1, ACTG1, ACTG2, ACTN1, ACTN2, ACTN4, ACTR3, ADAM10, ADSL, AGR2, AGR3, AGRN, AHCY, AHNAK, AKR1B10, ALB, ALDH16A1, ALDH1A1, ALDOA, ANXA1, ANXA11, ANXA2, ANXA2P2, ANXA4, ANXA5, ANXA6, AP2A1, AP2A2, APOA1, ARF1, ARF3, ARF4, ARF5, ARF6, ARHGDIA, ARPC3, ARPC5L, ARRDC1, ARVCF, ASCC3L1, ASNS, ATP1A1, ATP1A2, ATP1A3, ATP1B1, ATP4A, ATP5A1, ATP5B, ATP5I, ATP5L, ATP5O, ATP6AP2, B2M, BAIAP2, BAIAP2L1, BRI3BP, BSG, BUB3, C1orf58, C5orf32, CAD, CALM1, CALM2, CALM3, CAND1, CANX, CAPZA1, CBR1, CBR3, CCT2, CCT3, CCT4, CCT5, CCT6A, CCT7, CCT8, CD44, CD46, CD55, CD59, CD63, CD81, CD82, CD9, CDC42, CDH1, CDH17, CEACAM5, CFL1, CFL2, CHMP1A, CHMP2A, CHMP4B, CKB, CLDN3, CLDN4, CLDN7, CLIC1, CLIC4, CLSTN1, CLTC, CLTCL1, CLU, COL12A1, COPB1, COPB2, CORO1C, COX4I1, COX5B, CRYZ, CSPG4, CSRP1, CST3, CTNNA1, CTNNB1, CTNND1, CTTN, CYFIP1, DCD, DERA, DIP2A, DIP2B, DIP2C, DMBT1, DPEP1, DPP4, DYNC1H1, EDIL3, EEF1A1, EEF1A2, EEF1AL3, EEF1G, EEF2, EFNB1, EGFR, EHD1, EHD4, EIF3EIP, EIF3I, EIF4A1, EIF4A2, ENO1, ENO2, ENO3, EPHA2, EPHA5, EPHB1, EPHB2, EPHB3, EPHB4, EPPK1, ESD, EZR, F11R, F5, F7, FAM125A, FAM125B, FAM129B, FASLG, FASN, FAT, FCGBP, FER1L3, FKBP1A, FLNA, FLNB, FLOT1, FLOT2, G6PD, GAPDH, GARS, GCN1L1, GDI2, GK, GMDS, GNA13, GNAI2, GNAI3, GNAS, GNB1, GNB2, GNB2L1, GNB3, GNB4, GNG12, GOLGA7, GPA33, GPI, GPRC5A, GSN, GSTP1, H2AFJ, HADHA, hCG_1757335, HEPH, HIST1H2AB, HIST1H2AE, HIST1H2AJ, HIST1H2AK, HIST1H4A, HIST1H4B, HIST1H4C, HIST1H4D, HIST1H4E, HIST1H4F, HIST1H4H, HIST1H4I, HIST1H4J, HIST1H4K, HIST1H4L, HIST2H2AC, HIST2H4A, HIST2H4B, HIST3H2A, HIST4H4, HLA-A, HLA-A29.1, HLA-B, HLA-C, HLA-E, HLA-H, HNRNPA2B1, HNRNPH2, HPCAL1, HRAS, HSD17B4, HSP90AA1, HSP90AA2, HSP90AA4P, HSP90AB1, HSP90AB2P, HSP90AB3P, HSP90B1, HSPA1A, HSPA1B, HSPA1L, HSPA2, HSPA4, HSPA5, HSPA6, HSPA7, HSPA8, HSPA9, HSPD1, HSPE1, HSPG2, HYOU1, IDH1, IFITM1, IFITM2, IFITM3, IGH@, IGHG1, IGHG2, IGHG3, IGHG4, IGHM, IGHV4-31, IGK@, IGKC, IGKV1-5, IGKV2-24, IGKV3-20, IGSF3, IGSF8, IQGAP1, IQGAP2, ITGA2, ITGA3, ITGA6, ITGAV, ITGB1, ITGB4, JUP, KIAA0174, KIAA1199, KPNB1, KRAS, KRT1, KRT10, KRT13, KRT14, KRT15, KRT16, KRT17, KRT18, KRT19, KRT2, KRT20, KRT24, KRT25, KRT27, KRT28, KRT3, KRT4, KRT5, KRT6A, KRT6B, KRT6C, KRT7, KRT75, KRT76, KRT77, KRT79, KRT8, KRT9, LAMA5, LAMP1, LDHA, LDHB, LFNG, LGALS3, LGALS3BP, LGALS4, LIMA1, LIN7A, LIN7C, LOC100128936, LOC100130553, LOC100133382, LOC100133739, LOC284889, LOC388524, LOC388720, LOC442497, LOC653269, LRP4, LRPPRC, LRSAM1, LSR, LYZ, MAN1A1, MAP4K4, MARCKS, MARCKSL1, METRNL, MFGE8, MICA, MIF, MINK1, MITD1, MMP7, MOBKL1A, MSN, MTCH2, MUC13, MYADM, MYH10, MYH11, MYH14, MYH9, MYL6, MYL6B, MYO1C, MYO1D, NARS, NCALD, NCSTN, NEDD4, NEDD4L, NME1, NME2, NOTCH1, NQO1, NRAS, P4HB, PCBP1, PCNA, PCSK9, PDCD6, PDCD6IP, PDIA3, PDXK, PEBP1, PFN1, PGK1, PHB, PHB2, PKM2, PLEC1, PLEKHB2, PLSCR3, PLXNA1, PLXNB2, PPIA, PPIB, PPP2R1A, PRDX1, PRDX2, PRDX3, PRDX5, PRDX6, PRKAR2A, PRKDC, PRSS23, PSMA2, PSMC6, PSMD11, PSMD3, PSME3, PTGFRN, PTPRF, PYGB, QPCT, QSOX1, RAB10, RAB11A, RAB11B, RAB13, RAB14, RAB15, RAB1A, RAB1B, RAB2A, RAB33B, RAB35, RAB43, RAB4B, RAB5A, RAB5B, RAB5C, RAB6A, RAB6B, RAB7A, RAB8A, RAB8B, RAC1, RAC3, RALA, RALB, RAN, RANP1, RAP1A, RAP1B, RAP2A, RAP2B, RAP2C, RDX, REG4, RHOA, RHOC, RHOG, ROCK2, RP11-631M21.2, RPL10A, RPL12, RPL6, RPL8, RPLP0, the RPLP0 sample, RPLP1, RPLP2, RPN1, RPS13, RPS14, RPS15A, RPS16, RPS18, RPS20, RPS21, RPS27A, RPS3, RPS4X, RPS4Y1, RPS4Y2, RPS7, RPS8, RPSA, RPSAP15, RRAS, RRAS2, RUVBL1, RUVBL2, S100A10, S100A11, S100A14, S100A16, S100A6, S100P, SDC1, SDC4, SDCBP, SDCBP2, SERINC1, SERINC5, SERPINA1, SERPINF1, SETD4, SFN, SLC12A2, SLC12A7, SLC16A1, SLC1A5, SLC25A4, SLC25A5, SLC25A6, SLC29A1, SLC2A1, SLC3A2, SLC44A1, SLC7A5, SLC9A3R1, SMPDL3B, SNAP23, SND1, SOD1, SORT1, SPTAN1, SPTBN1, SSBP1, SSR4, TACSTD1, TAGLN2, TBCA, TCEB1, TCP1, TF, TFRC, THBS1, TJP2, TKT, TMED2, TNFSF10, TNIK, TNKS1BP1, TNPO3, TOLLIP, TOMM22, TPI1, TPM1, TRAP1, TSG101, TSPAN1, TSPAN14, TSPAN15, TSPAN6, TSPAN8, TSTA3, TTYH3, TUBA1A, TUBA1B, TUBA1C, TUBA3C, TUBA3D, TUBA3E, TUBA4A, TUBA4B, TUBA8, TUBB, TUBB2A, TUBB2B, TUBB2C, TUBB3, TUBB4, TUBB4Q, TUBB6, TUFM, TXN, UBA1, UBA52, UBB, UBC, UBE2N, UBE2V2, UGDH, UQCRC2, VAMP1, VAMP3, VAMP8, VCP, VIL1, VPS25, VPS28, VPS35, VPS36, VPS37B, VPS37C, WDR1, YWHAB, YWHAE, YWHAG, YWHAH, one or more in YWHAQ and YWHAZ.Described biomarker can be associated with the vesica film.

In embodiments, described biological sample comprises body fluid.Described body fluid can comprise peripheral blood, serum, blood plasma, ascites, urine, cerebrospinal fluid (CSF), phlegm, saliva, marrow, synovia, aqueous humor, amniotic fluid, earwax, milk, bronchoalveolar lavage fluid, seminal fluid, prostatic fluid, examine amber liquid or the liquid of ejaculating in advance, the women penetrates liquid, sweat, movement, hair, tear, capsule liquid, hydrothorax and ascites fluid, pericardial fluid, lymph liquid, the food gruel, chyle, bile, interstitial fluid, menses, purulence, sebum, vomitus, vaginal secretions, mucous membrane secretory product, just rare, pancreatic juice, nasal lavage fluid, the segmental bronchus aspirated liquid, blastochyle or Cord blood.Described biological sample can be, for example ight soil, blood or blood derivatives.Blood derivatives includes but not limited to blood plasma and serum.

In some embodiments, described biological sample comprises one or more microcapsule bubbles.In the described biological marking, one or more included biomarkers can be associated with described one or more microcapsule bubbles.When the described biological marking comprises multiple markers, some marks can be associated with vesica, and other mark is not associated.Perhaps, the whole marks in the described biological marking can be associated with described one or more microcapsule bubbles.

One or more surface antigens by described wedding agent identification can be one or more protein, for example, and the surface protein on described microcapsule bubble.Described one or more protein can comprise one or more in the listed protein of four transmembrane proteins, CD9, CD63, CD81, CD63, CD9, CD81, CD82, CD37, CD53, Rab-5b, annexin V, MFG-E8 or this paper table 3.

Described microcapsule bubble useful load can comprise one or more nucleic acid, peptide, protein, lipid, antigen, carbohydrate and/or proteoglycan.Described nucleic acid can comprise one or more DNA, mRNA, microRNA, snoRNA, snRNA, rRNA, tRNA, siRNA, hnRNA or shRNA.In one embodiment, described nucleic acid comprises one or more microRNAs or its combination that is selected from this paper table 6.

Several different methods of the present invention can be carried out external enforcement.

On the other hand, the invention provides the purposes of the reagent for implementing any means of the present invention.Described reagent can be used for diagnosis, prognosis or the treatment diagnosis of disease or imbalance (as prostate gland imbalance or colorectum imbalance).At a related aspect, the present invention also provides the test kit that comprises the reagent for implementing any means of the present invention.

In yet another aspect, the invention provides and comprise the composition that separates vesica.In one embodiment, described separation vesica comprises one or more biomarkers that are selected from this paper table 5.In another embodiment, described separation vesica comprises one or more biomarkers that are selected from this paper table 6.In another embodiment, described separation vesica comprises one or more biomarkers that are selected from this paper table 9-11.

Be incorporated to by reference

All publication, patents and patent applications of mentioning in this specification sheets all are incorporated herein by reference, open, the patent that its degree of quoting is independent as every portion or patent application all specifically and individually show to be incorporated to by reference.

The accompanying drawing summary

Fig. 1 (a)-(g) shows form, it has listed pedigree by cell/tissue, group relatively and the exemplary cancer of specific morbid state, and these cancers, grouping cell/tissue comparison and specific morbid state are had to specific antigen.In addition, described antigen can be biomarker.Described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Fig. 2 (a)-(f) shows form, it has listed pedigree by cell/tissue, group relatively and the exemplary cancer of specific morbid state, and these cancers, group cell/tissue comparison and specific morbid state are had to specific wedding agent.

Fig. 3 (a)-(b) is the form of having listed exemplary mammary cancer biomarker, and wherein said biomarker can be analyzed to produce the biological marking of the specific vesica of mammary cancer from the specific vesica of mammary cancer and to it.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Fig. 4 (a)-(b) is the form of having listed exemplary ovarian cancer biomarker, and wherein said biomarker can be analyzed to produce the specific biological marking of ovarian cancer from the specific vesica of ovarian cancer and to it.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Fig. 5 is the form of having listed the exemplary lung cancer biomarker, and wherein said biomarker can be analyzed to produce the specific biological marking of lung cancer from the specific vesica of lung cancer and to it.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Fig. 6 (a)-(d) is the form of having listed exemplary colorectal carcinoma biomarker, and wherein said biomarker can be analyzed to have produced the specific biological marking of colorectal carcinoma from the specific vesica of colorectal carcinoma and to it.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Fig. 7 has listed adenoma to be had to the form of specific exemplary biomarker to hyperplastic polyp, wherein said biomarker can be from adenoma to the specific vesica of hyperplastic polyp and it is analyzed.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Fig. 8 has listed for inflammatory bowel (IBD) normally having the form of specific exemplary biomarker, and wherein said biomarker can be from inflammatory bowel phase normal tissue is had specific vesica and it is analyzed.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Fig. 9 (a)-(c) has listed the form that has specific exemplary biomarker for the relative colorectal carcinoma of adenoma (CRC), and wherein said biomarker can be from for adenoma, colorectal carcinoma being had specific vesica and it is analyzed.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Figure 10 has listed the form that has specific exemplary biomarker for the relative CRC of IBD, and wherein said biomarker can be from for IBD, CRC being had specific vesica and it is analyzed.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Figure 11 (a)-(b) has listed the form that has specific exemplary biomarker for the relative Dukes CRC of Dukes CRC B C-D, and wherein said biomarker can be from for the relative Dukes CRC of Dukes CRC B C-D, having specific vesica and it is analyzed.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Figure 12 (a)-(d) is that the adenoma of having listed for low dysplasia has the form of specific exemplary biomarker to the adenoma of high grade dysplasia, and wherein said biomarker can the adenoma to high grade dysplasia have specific vesica and it is analyzed from the adenoma for low dysplasia.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Figure 13 (a)-(b) has listed the form that has specific exemplary biomarker for the relative Crohn's disease (CD) of ulcerative colitis (UC), and wherein said biomarker can be from for the relative CD of UC, having specific vesica and it is analyzed.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Figure 14 has listed the form that relatively normally has specific exemplary biomarker for hyperplastic polyp, and wherein said biomarker can be from relatively normally having specific vesica for hyperplastic polyp and it being analyzed.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Figure 15 has listed the form that relatively normally has specific exemplary biomarker for the adenoma with low dysplasia, and wherein said biomarker can relatively normally have specific vesica and it is analyzed from the adenoma for having low dysplasia.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Figure 16 has listed the form that relatively normally has specific exemplary biomarker for adenoma, and wherein said biomarker can be from relatively normally having specific vesica for adenoma and it being analyzed.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Figure 17 has listed the form that relatively normally has specific exemplary biomarker for CRC, and wherein said biomarker can be from relatively normally having specific vesica for CRC and it being analyzed.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Figure 18 is the form of having listed the specific exemplary biomarker of benign prostatic hyperplasia, and wherein said biomarker can and be analyzed it from the specific vesica of benign prostatic hyperplasia.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Figure 19 (a)-(c) is the form of having listed exemplary prostate cancer biomarker, and wherein said biomarker can and be analyzed to produce the biological marking of prostatic cancer specific to it from the vesica of prostatic cancer specific.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Figure 20 (a)-(c) is the form of having listed exemplary melanoma biomarker, and wherein said biomarker can be analyzed to produce the specific biological marking of melanoma from the specific vesica of melanoma and to it.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Figure 21 (a)-(b) is the form of having listed exemplary carcinoma of the pancreas biomarker, and wherein said biomarker can be analyzed to produce the specific biological marking of carcinoma of the pancreas from the specific vesica of carcinoma of the pancreas and to it.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Figure 22 is the form of having listed the specific exemplary biomarker of the cancer of the brain, and wherein said biomarker can be analyzed to produce the specific biological marking of the cancer of the brain from the specific vesica of the cancer of the brain and to it.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Figure 23 (a)-(b) is the form of having listed exemplary psoriatic biomarker, and wherein said biomarker can be analyzed to produce the specific biological marking of psoriatic from the specific vesica of psoriatic and to it.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Figure 24 (a)-(c) is the form of having listed exemplary cardiovascular disorder biomarker, and wherein said biomarker can be analyzed to produce the specific biological marking of cardiovascular disorder from the specific vesica of cardiovascular disorder and to it.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Figure 25 is the form of having listed the specific exemplary biomarker of hematologic malignancies, and wherein said biomarker can be analyzed to produce the specific biological marking of hematologic malignancies from the specific vesica of hematologic malignancies and to it.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Figure 26 (a)-(b) is the form of having listed the specific exemplary biomarker of B cell lymphocytic leukemia, and wherein said biomarker can be analyzed to produce the specific biological marking of B cell lymphocytic leukemia from the specific vesica of B cell lymphocytic leukemia and to it.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Figure 27 is the form of having listed B cell lymphoma and the specific exemplary biomarker of B cell lymphoma-DLBCL, and wherein said biomarker can and be analyzed it from B cell lymphoma and the specific vesica of B cell lymphoma-DLBCL.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Figure 28 is the form of having listed B cell lymphoma-DLBCL-germinal center sample, B cell lymphoma-DLBCL-activating B cell sample and the specific exemplary biomarker of B cell lymphoma DLBCL, and wherein said biomarker can and be analyzed it from B cell lymphoma-DLBCL-germinal center sample, B cell lymphoma-DLBCL-activating B cell sample and the specific vesica of B cell lymphoma-DLBCL.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Figure 29 is the form of having listed exemplary burkitt's lymphoma biomarker, and wherein said biomarker can be analyzed to produce the specific biological marking of burkitt's lymphoma from the specific vesica of burkitt's lymphoma and to it.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Figure 30 (a)-(b) is the form of having listed exemplary hepatocellular carcinoma biomarker, and wherein said biomarker can be analyzed to produce the specific biological marking of hepatocellular carcinoma from the specific vesica of hepatocellular carcinoma and to it.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Figure 31 is the form of having listed exemplary cervical cancer biomarker, and wherein said biomarker can and be analyzed it from the specific vesica of cervical cancer.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Figure 32 is the form of having listed exemplary carcinoma of endometrium biomarker, and wherein said biomarker can be analyzed to produce the specific biological marking of carcinoma of endometrium from the specific vesica of carcinoma of endometrium and to it.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Figure 33 (a)-(b) is the form of having listed exemplary head and neck cancer biomarker, and wherein said biomarker can be analyzed to produce the specific biological marking of head and neck cancer from the specific vesica of head and neck cancer and to it.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Figure 34 is the form of having listed exemplary inflammatory bowel (IBD) biomarker, and wherein said biomarker can be analyzed to produce the specific biological marking of IBD from the specific vesica of IBD and to it.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Figure 35 is the form of having listed exemplary diabetes biomarker, and wherein said biomarker can be analyzed to produce the specific biological marking of diabetes from the specific vesica of diabetes and to it.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Figure 36 is the form of having listed exemplary Barrett esophagus (Barrett ' s Esophagus) biomarker, and wherein said biomarker can be analyzed to produce the specific biological marking of Barrett esophagus from the specific vesica of Barrett esophagus and to it.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Figure 37 is the form of having listed exemplary fiber myalgia biomarker, and wherein said biomarker can and be analyzed it from the specific vesica of fibromyalgia.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Figure 38 is the form of having listed exemplary apoplexy biomarker, and wherein said biomarker can be analyzed to produce the specific biological marking of apoplexy from the specific vesica of apoplexy and to it.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Figure 39 is the form of having listed exemplary multiple sclerosis (MS) biomarker, and wherein said biomarker can be analyzed to produce the specific biological marking of MS from the specific vesica of MS and to it.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Figure 40 (a)-(b) is the form of having listed exemplary Parkinson's disease biomarker, and wherein said biomarker can be analyzed to produce the specific biological marking of Parkinson's disease from the specific vesica of Parkinson's disease and to it.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Figure 41 is the form of having listed exemplary rheumatosis biomarker, and wherein said biomarker can be analyzed to produce the specific biological marking of rheumatosis from the specific vesica of rheumatosis and to it.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Figure 42 (a)-(b) is the form of having listed exemplary Alzheimer biomarker, and wherein said biomarker can be analyzed to produce the specific biological marking of Alzheimer from the specific vesica of Alzheimer and to it.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Figure 43 is the form of having listed exemplary prion disease biomarker, and wherein said biomarker can be analyzed to produce the specific biological marking of prion disease from the specific vesica of prion disease and to it.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Figure 44 is the form of having listed exemplary Sepsis biomarker, and wherein said biomarker can be analyzed to produce the specific biological marking of Sepsis from the specific vesica of Sepsis and to it.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Figure 45 is the form of having listed exemplary chronic neuropathic pain model biomarker, and wherein said biomarker can and be analyzed it from the specific vesica of chronic neuropathic pain model.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Figure 46 is the form of having listed exemplary peripheral nerve pathologic pain biomarker, and wherein said biomarker can and be analyzed it from the vesica of peripheral nerve pathologic pain specific.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Figure 47 is the form of having listed exemplary schizophrenia biomarker, and wherein said biomarker can be analyzed to produce the specific biological marking of schizophrenia from the specific vesica of schizophrenia and to it.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Figure 48 is the form of having listed exemplary bipolar disorder or disease biomarker, and wherein said biomarker can be analyzed to produce the specific biological marking of bipolar disorder from the specific vesica of bipolar disorder and to it.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Figure 49 is the form of having listed exemplary dysthymia disorders biomarker, and wherein said biomarker can be analyzed to produce the specific biological marking of dysthymia disorders from the specific vesica of dysthymia disorders and to it.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Figure 50 is the form of having listed exemplary gastrointestinal stromal tumor (GIST) biomarker, and wherein said biomarker can be analyzed to produce the specific biological marking of GIST from the specific vesica of GIST and to it.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Figure 51 (a)-(b) is the form of having listed exemplary renal cell carcinoma (RCC) biomarker, and wherein said biomarker can be analyzed to produce the specific biological marking of RCC from the specific vesica of RCC and to it.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Figure 52 is the form of having listed exemplary liver cirrhosis biomarker, and wherein said biomarker can be analyzed to produce the specific biological marking of liver cirrhosis from the specific vesica of liver cirrhosis and to it.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Figure 53 is the form of having listed exemplary esophageal carcinoma biomarker, and wherein said biomarker can be analyzed to produce the specific biological marking of the esophageal carcinoma from the specific vesica of the esophageal carcinoma and to it.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Figure 54 is the form of having listed exemplary cancer of the stomach biomarker, and wherein said biomarker can be analyzed to produce the specific biological marking of cancer of the stomach from the specific vesica of cancer of the stomach and to it.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Figure 55 is the form of having listed exemplary autism biomarker, and wherein said biomarker can be analyzed to produce the specific biological marking of autism from the specific vesica of autism and to it.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Figure 56 is the form of having listed exemplary organ rejection response biomarker, and wherein said biomarker can be analyzed to produce the specific biological marking of organ rejection response from the specific vesica of organ rejection response and to it.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Figure 57 is the form of having listed exemplary methicillin-resistant staphylococcus aureus biomarker, and wherein said biomarker can be analyzed to produce the specific biological marking of methicillin-resistant staphylococcus aureus from the specific vesica of methicillin-resistant staphylococcus aureus and to it.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Figure 58 is the form of having listed example frangible patch biomarker, and wherein said biomarker can be analyzed to produce the specific biological marking of vulnerable plaque from the specific vesica of vulnerable plaque and to it.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, (for example outer genetic modification or posttranslational modification) sudden change or that modify.

Figure 59 (a)-(i) is the form of having listed exemplary gene fusion, and wherein said gene fusion can be analyzed from vesica or by vesica.Described gene fusion can be biomarker, and can be exist or non-existent, express not enough or cross and express, or (for example outer genetic modification or posttranslational modification) of modifying.

The form that Figure 60 (a)-(b) is gene and relevant miRNA thereof, wherein said gene (for example mRNA of this gene), its relevant miRNA or their arbitrary combination can be as one or more biomarkers that can be obtained by the vesica analysis.In addition, described one or more biomarkers can be exist or be non-existent, express not enough or cross and express, sudden change or modify.

Figure 61 A has described and has identified that the biological marking that comprises nucleic acid is to characterize the method for phenotype.Figure 61 B has described the biological marking of evaluation vesica or vesica colony to characterize the method for phenotype.

Figure 62 describes in detail the protein on vesica is screened to obtained result, and described protein can be used as the biomarker of described vesica.Antibody for described protein can be used as wedding agent.The example of the protein of the biomarker through being accredited as vesica comprises Bcl-XL, ERCC1, keratin 15, CD81/TAPA-1, CD9, epithelial specific antigen (ESA) and mastocyte Chymotrypsin.Described biomarker can be to exist or non-existent in vesica or on vesica, express not enough or cross and express, sudden change or modify, and can be used for the sign situation.

Figure 63 describes the method that characterizes phenotype by the biological marking of assessment vesica in detail.Figure 63 A is the sketch that is coated with the planar substrates of capture antibody, and described capture antibody is caught the vesica of expressing described protein.Described capture antibody is for vesicle protein matter (it has or do not have specificity to the vesica (" disease vesica ") that is derived from sick cell).Described detection antibody is combined with the vesica of catching and fluorescent signal is provided.Described detection antibody can detect the antigen relevant to vesica usually, or the detection antigen relevant to cell source or disease (as cancer).Figure 63 B is the sketch that is coated with the pearl of capture antibody, and described capture antibody is caught the vesica of expressing described protein.Described capture antibody is for vesicle protein matter (it has or do not have specificity for the vesica that is derived from sick cell (" disease vesica ")).Described detection antibody is combined with the vesica of catching and fluorescent signal is provided.Described detection antibody can detect the antigen relevant to vesica usually, or the detection antigen relevant to cell source or disease (as cancer).The example that Figure 63 C is screening scheme, it can be implemented in multiple mode by using the pearl as shown in Figure 63 B.Figure 63 D shows and catches or detect vesica to characterize the explanatory view of phenotype.Figure 63 E shows for assessment of the vesica useful load to characterize the schematic diagram of phenotype.

The schematic diagram that Figure 64 is protein expression mode.Usually, different protein not on average or equably is distributed on the shell of vesica.The specific protein of vesica is usually more common, and the protein of cancer specific is more rare.Use the protein of more common low cancer specific can more easily complete catching of vesica, and the protein of cancer specific can be for detection of in the stage.

Figure 65 describes the computer system that can use in exemplary more of the present invention in detail.

Figure 66 A-B has described the scanning electron photomicrograph (SEM) of the pearl of EpCam coupling, and wherein said pearl hatches together with the VcaP vesica.

Figure 67 describes in detail and uses detection to describe the method for result from the method based on pearl of experimenter's vesica.Figure 67 A is for single patient, uses the pearl quantity of the screening scheme described in Figure 63 B and the figure of strength of signal, catches pearl for wherein～100 and respectively catches/detect binding analysis for each patient.For given patient, Output rusults has shown the quantity of the pearl detected of relative signal intensity.The quantity of the pearl caught under given intensity is the indication of vesica with what kind of frequency detection of expression protein under described intensity.For given pearl, the intensity of signal is stronger, detects protein expression more.Figure 67 B is for by normal patient is combined in a curve cancer patients is combined in another curve, and uses biometrics to analyze the normalized figure that distinguishes described curve and obtain.To the data normalization that obtained by each individuality to consider the difference by the quantity of the pearl that detecting instrument was read, these data are added and, and then sample sizes different in each colony is considered in normalization method.

Figure 68 describes the biological marking that illustrates prostate cancer in detail.The serve as reasons histogram of the intensity level that uses the multiple test of microballoon platform and collect of Figure 68 (A), wherein integument CD63 antibody function, hatch with the vesica by patient's blood plasma purifying, then uses the EpCam antibody labeling of phycoerythrin (PE) coupling.The post of darker shade (indigo plant) means 12 normal subjectses' colony, and the post of more shallow shade (green) derives from 73 phase patients with prostate cancer.Figure 68 (B) carries out normalized figure according to Figure 67 is described to each histogram shown in Figure 68 (A).For patients with prostate cancer sample and normal specimens, be distributed as the Gauss curve fitting to the intensity level of the microballoon result from Figure 68 (A).The example that Figure 68 (C) is one of biological marking of the prostate cancer shown in Figure 68 (B), CD63 is to the biological marking (figure of top) of CD63, and wherein CD63 is as detection agent and capture antibody.3 results that illustrate the flow cytometry on 3 kinds of prostate cancer cell lines (VCaP, Lncap and 22RV1) of below.The point of sea line top has meaned to catch the pearl of the vesica (comprising B7H3) with CD63.The pearl on vertical line right side has meaned to catch the pearl of the vesica (having PSMA) with CD63.These pearls on online top and line right side have 3 kinds of all antigens.CD63 is the surface protein relevant to vesica, and PSMA is the surface protein relevant to prostatic cell, and B7H3 is the surface protein relevant to invasive cancer (being particularly prostate cancer, ovarian cancer and nonsmall-cell lung cancer).All these 3 kinds of antigens combine and identify the vesica from the cancer prostatic cell.The prostate cancer vesica of most of CD63 of expression also has prostate specific membrane antigen PSMA and B7H3 (participate in regulate tumor cell migration and invasion, and be the indication of invasive cancer and clinical effectiveness).The form that Figure 68 (D) is the prostate cancer vesica.The illustrating of top used CD63, CD9 and CD81 to be caught the result with mark with multiple combination.Nearly all point all is positioned at right upper quadrant, shows that these 3 kinds of marks are height couplings.Next column has described that to use B7H3 to catch and use CD63 and PSMA labeled cell be the result of vesica.VCaP and 22RV1 show, the most of vesicas that use B7H3 to catch also have CD63, and have two kind of groups, that is, have the colony of PSMA and do not have the colony of PSMA.Because LNcap does not have a large amount of vesica that comprises B7H3 (not having the point that has in a large number CD63), the existence of B7H3 can be the indication of invasive cancer degree.LnCap is the similar clone of early prostate cancer.

Figure 69 has illustrated the biological marking of colorectal carcinoma.(A) described the histogram of the intensity level of being collected by the various multiple test of having used the microballoon platform, wherein used capture antibody to the pearl functionalization, it has been hatched with together with vesica from patient's blood plasma purifying, then with detecting antibody labeling.Darker shade post (indigo plant) means from normal colony, and more shallow shade post (green) is from colorectal cancer patients.(B) show the normalization method figure of each histogram shown in (A).(C) describe the histogram of the intensity level of being collected by multiple test, wherein hatched with together with vesica by patient's blood plasma purifying by the pearl of CD66 antibody (capture antibody) functionalization, then used EpCam antibody (detection antibody) mark with the PE coupling.Red colony is from 6 normal experimenters, and green colony is from 21 colorectal cancer patients.The difference of the pearl number that the data normalization that will be obtained by each individuality is detected with consideration, these data are added together, and then normalization method is to consider sample numbers different in each colony.

Figure 70 shows that the Multiple detection agent can increase signal.(A) when vesica is marked with the antibody of multiple and prostate specific PE coupling, the meta intensity level is plotted as the function from the purifying concentration of VCaP clone.The vesica of catching with EpCam (figure in left side) or PCSA (figure on right side) has been listed and by detecting the multiple proteins that antibody was detected in the right side of each figure.In both cases, the combination of CD9 and CD63 has all obtained exceeding the optimum signal increase (bottom diagram describes to increase percentage) of background.About 200% the percentage that the combination of CD9 and CD63 obtains exceeding background increases.(B) further show the multiplexing detection that has improved the vesica in prostate cancer cell source of prostate cancer/prostate gland vesica Specific marker.When the antibody that uses multiple prostate specific PE coupling carries out mark, the meta intensity level is plotted as the function from the purifying concentration of VCaP clone.Described the vesica of catching with PCSA (figure in left side) and EpCam (figure on right side).In both cases, the combination of B7H3 and PSMA has all obtained exceeding the optimum signal increase of background.

Figure 71 has illustrated and has used CD63 detection agent and CD63 to catch by the stage for the biological marking of the colorectal carcinoma of colorectal carcinoma.The intensity histogram derives from that catch and the vesica that use CD63 coupling PE mark of the pearl of using the CD63 coating.6 patients (A) are arranged in control group, and I is interim 4 patients (B), and II is interim 5 patients (C), and III is interim 8 patients (D), and IV is interim that 4 patients (E) are arranged.The variation of the pearl quantity that the data that obtained by each individuality are detected with consideration through normalization method, these data are added together, and then normalization method is to consider the different sample numbers (F) in each colony.

Figure 72 has illustrated and has used EpCam detection agent and CD9 to catch for the biological marking of the colorectal carcinoma of the colorectal carcinoma by the stage.The intensity histogram derives from that catch and the vesica that use the EPCam mark of the pearl of using the CD9 coating.Wherein, in control group (A), I phase (B), II phase (C), III phase (D) and VI phase (E), the patient is arranged.The variation of the pearl quantity that the data that obtained by each individuality are detected with consideration with normalization method, these data are added together, and then normalization method is to consider the different sample numbers (F) in each colony.

Figure 73 has illustrated that (A) used sensitivity, specificity and the level of confidence of antibody (listing as detecting and capture antibody) the detection prostate cancer for listed protein.CD63, CD9 and CD81 are general mark, and EpCam is cancer markers.Single result is depicted in (B), for EpCam for CD63, the degree of confidence with 99%, cancer patients's sample of 100% (n=8) is different from the broad sense normal distribution, and the degree of confidence with 99%, the normal patient sample of 77% (n=10) is different from the broad sense normal distribution; (C) for CD81 for CD63, the degree of confidence with 99%, cancer patients's sample of 90% (n=5) is different from the broad sense normal distribution, the degree of confidence with 99%, the normal patient sample of 77% (n=10) is different from the broad sense normal distribution; (D) for CD63 for CD63, the degree of confidence with 99%, cancer patients's sample of 60% (n=5) is different from the broad sense normal distribution, the degree of confidence with 99%, the normal patient sample of 80% (n=10) is different from the broad sense normal distribution; (E) for CD9 for CD63, the degree of confidence with 99%, cancer patients's sample of 90% (n=5) is different from the broad sense normal distribution, the degree of confidence with 99%, the normal patient sample of 77% (n=10) is different from the broad sense normal distribution.

Figure 74 has illustrated that (A) used sensitivity and the level of confidence of antibody (listing as detecting and capture antibody) the detection colorectal carcinoma for listed protein.CD63 and CD9 are general mark, and EpCam is cancer markers, and CD66 is the colon mark.Single result is depicted in (B), for EpCam for CD63, the degree of confidence with 99%, cancer patients's sample of 95% (n=20) is different from the broad sense normal distribution, degree of confidence with 99%, 100% (n=6) normal patient sample is different from the broad sense normal distribution; (C) for EpCam for CD9, the degree of confidence with 99%, cancer patients's sample of 90% (n=20) is different from the broad sense normal distribution, the degree of confidence with 99%, 77% (n=6) normal patient sample is different from the broad sense normal distribution; (D) for CD63 for CD63, the degree of confidence with 99%, cancer patients's sample of 60% (n=20) is different from the broad sense normal distribution, the degree of confidence with 99%, the normal patient sample of 80% (n=6) is different from the broad sense normal distribution; (E) for CD9 for CD63, the degree of confidence with 99%, cancer patients's sample of 90% (n=20) is different from the broad sense normal distribution, the degree of confidence with 99%, the normal patient sample of 77% (n=6) is different from the broad sense normal distribution; (F) for CD66 for CD9, the degree of confidence with 99%, cancer patients's sample of 90% (n=20) is different from the broad sense normal distribution, the degree of confidence with 99%, the normal patient sample of 77% (n=6) is different from the broad sense normal distribution.

Figure 75 has illustrated by assessment TMPRSS2-ERG and has expressed and use EpCam catching the prostate cancer cell source vesica from blood plasma.(A) the VCAP purifying vesica of gradual change amount is mixed in normal plasma.The Dynal pearl that use has EPCAM antibody or the contrast of its isotype separates vesica.Separation is from the RNA of vesica, and the expression of using qRT-PCR measurement TMPRSS2:ERG to merge transcript.(B) vesica of VcaP purifying is incorporated in normal plasma, then hatches together with the Dynal magnetic bead that is coated with EpCam or isotype control antibodies.By direct isolation of RNA on the Dynal pearl.Use the equal-volume RNA from each sample to carry out RT-PCR and the analysis of Taqman subsequently.(C) cycle threshold (CT) difference of SPINK1 and GAPDH transcript between the 22RV1 vesica that uses EpCam and IgG2 isotype negative control pearl to catch.The CT value is higher shows that the expression of transcript is lower.

Figure 76 has illustrated the microRNA that front 10 species diversity between VCaP prostate cancer cell source vesica and normal plasma vesica are expressed.By ultracentrifugation, then by RNA, separate and obtain VCAP clone vesica and from the vesica of normal plasma.Use qRT-PCR to analyze and obtain the microRNA spectrum.Prostate cancer cell line source vesica have higher level (low CT value) shown in microRNA, described at histogram.

Figure 77 has described the histogram of the miR-21 expression of using the CD9 pearl to catch.By deriving from the 1ml blood plasma of patients with prostate cancer, the LNCaP of 250ng/ml or the Dynal pearl of normal purifying vesica and CD9 coating, hatch.Isolation of RNA in described pearl and pearl supernatant liquor.In addition, a duplicate samples (#6) is not caught with for relatively.The expression of using qRT-PCR to measure miR-21, and the mean CT-number of each sample relatively.CD9 catches the detection that has improved miR-21 in the prostate cancer sample.

Figure 78 has described the histogram of the miR-141 expression of using the CD9 pearl to catch.This experiment is implemented according to Figure 77, the expression of wherein using qRT-PCR to measure miR-141, and be not miR-21.

Figure 79 shows and uses the trapping agent of CD9, CD63, CD81, PSMA, PCSA, B7H3 and EpCam the vesica detection of biological mark CD9, the CD81 that separate from sample (#126) and the figure of CD63 (A-D) or B7H3 and EpCam (E-H), described vesica is used the 500 μ l post (Millipore with 100kDa MWCO, Billerica, MA) (A, E), there is the 7ml post (Pierce of 150kDa MWCO

rockford, IL) (B, F), there is the 15ml post (Millipore, Billerica, MA) (C, G) of 100kDa MWCO or there is the 20ml post (Pierce of 150kDa MWCO

rockford, IL) (D, H) separation.

Figure 80 shows and uses the figure that the trapping agent of CD9, CD63, CD81, PSMA, PCSA, B7H3 and EpCam is detected biomarker CD9, the CD81 of the vesica that separates from sample (#342) and CD63 (A-D) or B7H3 and EpCam (E-H), described vesica is used the 500 μ l post (Millipore with 100kDa MWCO, Billerica, MA) (A, E), there is the 7ml post (Pierce of 150kDa MWCO

rockford, IL) (D, H) separation.

Figure 81 shows at a kind of sample (#126) and (A-C) uses the figure that the trapping agent of CD9, CD63, CD81, PSMA, PCSA, B7H3 and EpCam is detected biomarker CD9, the CD81 of vesica and CD63 or B7H3 and EpCam in relatively another kind of sample (#117) contrast (D-F), and it has used the 7ml post (Pierce with 150kDa MWCO

rockford, IL) (A, D), the 15ml post (Millipore, Billerica, MA) (B, E) with 100kDa MWCO or the 20ml post (Pierce of 150kDa MWCO

rockford, IL) (C, F).

Figure 82 is the figure that has shown the detection of biomarker CD9, CD63 and CD81, and it has used trapping agent A) CD9, B) PCSA, C) PSMA and D) EpCam.Vesica separates from control sample (healthy sample) and prostate cancer sample, II phase prostate cancer (PCa) sample.Separate and compare with the ultracentrifugation of vesica, by using the filter separation method based on post, improved the separation between PCa and contrast.

Figure 83 has described separation and has used the comparison between ultracentrifugation and the method based on strainer from the detection level of the various biomarkers of the vesica of patient's sample (#126), the described method based on strainer has been used the 500 μ l post (Millipore with 100kDa weight shutoff value (MWCO), Billerica, MA).Described figure has described A) sample of ultracentrifugation purifying; B) Microcon sample; C) ultracentrifugation purification of samples and 10ug Vcap and D) there is the Microcon sample of 10ug Vcap.The trapping agent used is CD9, CD63, CD81, PSMA, PCSA, B7H3 and EpCam, and CD9, CD81 and CD63 detected.

Figure 84 has described separation and has used the comparison between ultracentrifugation and the method based on strainer from the detection level of the various biomarkers of the vesica of patient's sample (#342), the described method based on strainer has been used the 500 μ l post (Millipore with 100kDa MWCO, Billerica, MA).Described figure has described A) sample of ultracentrifugation purifying; B) Microcon sample; C) ultracentrifugation purification of samples and 10ug Vcap and D) there is the Microcon sample of 10ug Vcap.The trapping agent used is CD9, CD63, CD81, PSMA, PCSA, B7H3 and EpCam, and CD9, CD81 and CD63 detected.

Figure 85 describes in detail and uses the vesica that MoFlo XDP carries out separate and identify.

Figure 86 A-86D describes the airflow classification of vesica in blood plasma in detail.Figure 86 A shows detection and the sorting to the positive vesica of PCSA in patients with prostate cancer blood plasma.Figure 86 B shows detection and the sorting to the positive vesica of CD45 in normal and patients with prostate cancer blood plasma.Figure 86 C shows detection and the sorting to the positive vesica of CD45 in normal and plasma of breast cancer patients.Figure 86 D shows detection and the sorting to the positive vesica of DLL4 in normal and patients with prostate cancer blood plasma.

Figure 87 is the schematic diagram that detects vesica in sample, wherein uses existence or the level of the desirable vesica of microballoon Platform evaluation.Figure 87 A is used the filter method based on post to separate the schematic diagram of vesica from blood plasma, and the vesica of wherein said separation is used the microballoon platform to be assessed subsequently.Figure 87 B causes the schematic diagram of vesica mould contracting due to the high speed centrifugation such such as ultracentrifugation.Figure 87 C is the schematic diagram that uses laser detection to be detected the vesica that is incorporated into microballoon.

Figure 88 A describes the ability that the biological marking of vesica is distinguished normal prostatic and PCa sample in detail.Cancer markers comprises EpCam and B7H3.General vesica mark comprises CD9, CD81 and CD63.The prostate specific mark comprises PCSA.It is found that described test has 96% sensitivity and 95% specificity for PCa contrast normal specimens.Figure 88 B shows the average fluorescent strength (MFI) of the vesica marker of Figure 88 A in normal and patients with prostate cancer on Y-axis.

The sensitivity of the improvement of Figure 89 A vesica analysis contrast of the present invention conventional P Ca test.Figure 89 B describes the specificity of the improvement of vesica analysis contrast conventional P Ca of the present invention test in detail.

Figure 90 describes in detail and uses CD63 to the differentiation with normal and PCa sample to the BPH sample.

Figure 91 describes the ability that the biological marking of vesica is distinguished normal prostatic and PCa sample in detail.Cancer markers comprises EpCam and B7H3.General vesica mark comprises CD9, CD81 and CD63.The prostate specific mark comprises PCSA.Normal and BPH sample has 98% sensitivity and 84% specificity for PCa contrast to it is found that described test.

Figure 92 describes the specificity of improving for the vesica analysis contrast traditional test of the present invention of PCa in detail, even in the situation that comprise the BPH sample.

Figure 93 describes the ROC curve of vesica analysis contrast conventional P Ca of the present invention test in detail.

Figure 94 shows general vesica (as vesica " MV ") level, prostate specific MV and has the dependency between the level of MV of cancer markers.

Figure 95 describes remarkable different vesica mark between PCa and normal specimens in detail.

Figure 96 is A) schematic diagram that the vesica prostate cancer is analyzed, it has produced decision tree B), C), D) with whether positive for prostate cancer for determining sample.

Figure 97 A shows according to described decision tree and analyzes for the vesica detection of prostate cancer the result detected with respect to the PSA level of using raising.Figure 97 B shows the vesica for the prostate cancer cohort of 933 routine PCa and non-PCa patient's sample carried out according to described decision tree and detects the result of analyzing.Figure 97 C shows the ROC curve corresponding to the data shown in Figure 97 B.

Figure 98 describes the MFI threshold value of using cluster analysis to set prostate cancer vesica biomarker in detail.A) raw data of 149 routine samples and log translation data.Described raw data is mapped in left hurdle and described translation data is mapped in right hurdle.B) use the log translation data to carry out the cluster analysis of PSMA vs B7H3 as input.Annulus (normally) and * (cancer) shown two clusters finding.Hollow large circle has shown the point that is used as cluster centre.Blue line has shown the cutoff of selecting for each parameter.C) cluster analysis of the PCSA vs B7H3 that use log translation data is carried out as input.Annulus (normally) and * (cancer) shown two clusters finding.Hollow large circle has shown the point as cluster centre.Blue line has shown the cutoff of selecting for each parameter.D) cluster analysis of the PSMA vs PCSA that use log translation data is carried out as input.Annulus and * shown two clusters finding.Red hollow large circle has shown the point as cluster centre.Blue line shows the cutoff of selecting for each parameter.E) will be at B-D) in definite threshold application in the larger data set that comprises 313 routine samples, and produced 92.8% sensitivity and 78.7% specificity.

Figure 99 is to show the average fluorescent strength (MFI) of the vesica in assessment prostate cancer (cancer) and normal (normally) sample on the y axle.The vesicle protein biomarker indicates on the x axle, and it comprises CD9, PSMA, PCSA, CD63, CD81, B7H3, IL-6, OPG-13 (also referred to as OPG), IL6R, PA2G4, EZH2, RUNX2, SERPINB3 and EpCam from left to right.

Figure 100 describes the differentiation of the relative III of the BPH phase PCa that uses antibody array to carry out in detail.

Figure 101 describes the miR-145 level of separating in the vesica of contrast and PCa sample in detail.

Figure 102 A-102B describes miR-107 (Figure 102 A) and miR-574-3p (Figure 102 B) level of separating in the vesica of contrast (non-PCa) and prostate cancer sample in detail, as indicated on X-axis.Use Taqman to analyze in the vesica separated and detect miR.This figure below shows the P value.Y-axis shows the copy number of the miR detected.In Figure 102 B, will be excluded in outside analysis from two outlier samples of various kinds product group, described outlier sample has the copy number of super sample deviation far away.

Figure 103 A-103D describes the level of separating miR-141 (Figure 103 A), miR-375 (Figure 103 B), miR-200b (Figure 103 C) and miR-574-3p (Figure 103 D) in the vesica of transitivity (M1) and non-metastatic (MO) prostate cancer sample in detail.MiR in the vesica that use Taqman analyzing and testing is separated.

Figure 104 A-104B describes in detail and uses miR-107 and miR141 to confirm the false negative from the Diagnosis of prostate cancer based on vesica.Figure 104 A describes the analysis of miR in the use vesica in detail false negative is converted to the schematic diagram of true positives, has improved thus sensitivity.Figure 104 B describes the analysis of miR in the use vesica in detail false positive is converted into to the schematic diagram of true negative, improves thus specificity.The normalization method level of miR-107 (Figure 104 C) and miR-141 (Figure 104 D) illustrates for the false positive (FP) of the true negative (TN) of the true positives (TP) of described vesica diagnositc analysis gained, described vesica diagnositc analysis gained, described vesica diagnositc analysis gained and the false negative (FN) of described vesica diagnositc analysis gained on Y-axis.

Figure 105 A-105F describe in detail suffer from or do not suffer from PCa and PSA >=or<column diagram that hsa-miR-432 (Figure 105 A), hsa-miR-143 (Figure 105 B), hsa-miR-424 (Figure 105 C), hsa-miR-204 (Figure 105 D), hsa-miR-581f (Figure 105 E) and hsa-miR-451 (Figure 105 F) in the patient of 4.0ng/ml raises.MiR in the vesica that use Taqman analyzing and testing is separated.Taqman analyzes the miR level detected and shows on Y-axis.X-axis shows four sample sets.From left to right, " contrasting non-" is the control patients of PSA >=4.0; " contrast is " is the control patients of PSA<4.0; " ill non-" is the patients with prostate cancer of PSA >=4.0; And " ill be " is the patients with prostate cancer of PSA<4.0.

Figure 106 describes in detail and is separating microRNA miR-29a in the vesica of prostate cancer (PCa) and the plasma sample that contrasts and the level of miR-145.

Figure 107 describes the slab design layout that microballon is analyzed in detail.

Figure 108 A-D describes the ability of distinguishing colorectal carcinoma (CRC) and the various different capture antibodies of the vesica of normal specimens for catching in detail.Figure 108 A has illustrated that the multiple of the capture antibody antigen (X-axis) of the relative normal specimens of CRC vesica sample changes (Y-axis), and it is measured according to antibody array.Figure 108 B is similar with it, and difference is according to shown in legend, and Y-axis is the meta fluorescence intensity in CRC and normal specimens.Figure 108 C is similar to Figure 108 B, and it implements on other sample sets.Figure 108 D shows and uses CD24 as the colon mark, and TROP2 is as cancer markers, and the analysis carried out as general vesica mark of four transmembrane protein CD9, CD63 and CD81.

Thereby Figure 109 A-H describes in detail by using TMEM211 and/or CD24 to detect vesica and detect CRC in plasma sample.Figure 109 A describes the ROC tracing analysis of the vesica analysis of the present invention of using biomarker TMEM211 to carry out in detail.Figure 109 B describes the ROC tracing analysis of the vesica analysis of the present invention of using biomarker CD24 to carry out in detail.Figure 109 C describes the analysis for normal, as to suffer from colorectal carcinoma (CRC) experimenter and the person's of obscuring vesica analytical procedure of the present invention in detail.Figure 109 D describes in detail and in follow-up study, uses biomarker TMEM211 for vesica sample analysis normal, that suffer from experimenter He the person of obscuring of colorectal carcinoma (CRC).Figure 109 E describes the ROC tracing analysis of the vesica analysis of the present invention of using biomarker TMEM211 to carry out in detail.Figure 109 F-109H describes the result of the other research of carrying out from the patient's cohort with expansion in detail.In Figure 109 F, the meta fluorescence intensity (MFI) of TMEM211 illustrates on X-axis, and the MFI of CD24 illustrates on Y-axis.TMEM211 and CD24 individually for the result of distinguishing various inhomogeneity samples respectively shown in Figure 109 G and Figure 109 H.

Figure 110 describes TaqMan low density array (TLDA) the miRNA card comparison of colorectal carcinoma (CRC) clone to normal vesica in detail.CRC clone marks on the figure right side.Y-axis has shown that the multiple that CRC clone is compared with normal control in expression changes.The miRNA monitored marks on X-axis, and be miR-548c-5p, miR-362-3p from left to right, miR-422a, miR-597, miR-429, miR-200a and miR-200b.For each miR, cylindricality from left to right is corresponding to clone LOVO, HT29, SW260, COLO205, HCT116 and RKO.These miRNA do not cross and express in normal or melanoma cell.

Figure 111 A describes in detail and uses miR92 and the miR491 differentiation to normal and CRC sample.Figure 111 B describes in detail and uses miR92 and the miR21 differentiation to normal and CRC sample.Figure 111 C describes the multiplexing to differentiation normal and the CRC sample of use miR92, miR21, miR9 and miR491 in detail.

Figure 112 describes the KRAS order-checking in colorectal carcinoma (CRC) clone and patient's sample in detail.Sample comprises available from clone (B) or the genomic dna that obtains from the tissue sample from described patient (D), or available from from described clone (A), come off or from the cDNA of the RNA useful load in vesica the plasma sample of described patient (C).

Figure 113 describes the distinguishing CRC by detecting from the TMEM211 in the microcapsule bubble of plasma sample and MUC1 in detail.X-axis (MUC1) and Y-axis (TMEM211) are corresponding to the meta fluorescence intensity (MFI) of detected vesica in described sample.Horizontal and vertical lines is respectively for TMEM211 and MUC1 and detect the MFI threshold value of CRC.

Figure 114 A describes the multiple that exceeds normal value that is described in the biomarker detected in patient with breast cancer's sample (n=10) or normal control (that is, without mammary cancer) in detail to be changed.Vesica in the antibody capture plasma sample of the antigen that indicates of use mooring on pearl.Use detects the vesica of catching for the traget antibody of four transmembrane protein CD9, CD63 and CD81.It is the multiple variation of the vesica that detects in the described mammary cancer sample meta fluorescence intensity (MFI) of comparing with normal specimens that multiple on Y-axis changes.Figure 114 B describes the level of the various different biomarkers that detect in the vesica that is derived from breast cancer cell MCF7, T47D and MDA in detail.T47D and MDA are metastatic cell systems.

Figure 115 A describes the multiple of comparing with normal specimens from the various different biomarkers in the membrane vesicle of lung cancer sample in detail to be changed, and it uses the antibody for marked vesica antigen to be detected.The black post is the ratio of lung cancer sample and normal specimens.The white post is the ratio of non-lung cancer sample and normal specimens.The data that underscore marks are shown in Figure 115 B.Figure 115 B has illustrated and has used the membrane vesicle fluorescence level for the antibody test of indicated vesica antigen.Fluorescence level is the mean value from following sample: normal (white), non-lung cancer sample (grey) and lung cancer sample (black) by stages.Figure 115 C shows the meta fluorescence intensity (MFI) of the vesica detection of using EPHA2 (i), CD24 (ii), EGFR (iii) and CEA (iV) to carry out in the sample from patients with lung cancer and normal control.Figure 115 D and Figure 115 E have provided the average fluorescent strength (MFI) of the vesica detected in the sample from lung cancer and normal (non-lung cancer) experimenter on Y-axis.Capture antibody marks along X-axis.

Figure 116 has provided and has used the capture antibody for detection of vesica that the marks decision tree with detection of lung cancer.

Figure 117 A describes in the vesica that is derived from blood plasma vesica level with respect to the CD81 mark of circulating tumor cell (CTC) in detail according to tumour cell (CTC).Measured for the vesica of collecting from patient's (" CTC " samples of the 14 routine leftmost sides) and normal plasma (4 routine rightmost side sample) with CD81 and the CTC counted, the vesica of measuring with CD81 and CTC has been counted.Figure 117 B describes in the vesica that is derived from EpCAM+ blood plasma the miR-21 copy number with respect to CTC in detail.Show patient's sample (" CTC " samples of the 15 routine leftmost sides) and normal specimens (" normally " samples of the 7 routine rightmost sides).By the assessment of the miR-21qRT-PCR from RNA copy number, described RNA extracts from the vesica that comes from EpCAM+ blood plasma.The CTC counting is available from same sample.

Figure 118 A-118C describes in detail from the vesica level in patient with breast cancer's blood plasma, and it uses the antibody for CD31 (Figure 118 A), DLL4 (118B) and CD9 (Figure 118 C) to be detected exhaust the positive vesica of CD31+ from described sample.

Figure 119 describes the detection to the tissue factor (TF) in the vesica from normal (non-cancer) plasma sample, mammary cancer (BCa) plasma sample and prostate cancer (PCa) plasma sample in detail.Use the anti-tissue factor antibodies of mooring on microballoon to catch the vesica in plasma sample.Use detects the vesica of catching for the traget antibody of four transmembrane protein CD9, CD63 and CD81.

Figure 120 A-C shows the epitope epitope mapping that uses anti-TMEM211 rabbit polyclonal antibody to carry out.Described antibody is tested described antibody for the peptide overlapping from a series of friendships of TMEM211.Figure 120 A shows the combination to described peptide with anti-TMEM211 rabbit polyclonal antibody and goat anti-rabbit igg HRP secondary antibody.Figure 120 B shows to contrast the combination to described peptide of rabbit polyclonal antibody and goat anti-rabbit igg HRP secondary antibody.Figure 120 C shows the result to the combination of described peptide with anti-TMEM211 rabbit polyclonal antibody and goat anti-mouse IgG HRP secondary antibody.

Figure 121 A-C shows the epitope mapping that uses anti-B7H3 (B7-H3) rat monoclonal antibody to carry out.Described antibody is for a series of overlapping peptide test of B7H3.Figure 121 A shows the combination to described peptide with anti-B7H3 rat monoclonal antibody and the mouse IgG HRP of goat Chinese People's Anti-Japanese Military and Political College secondary antibody.Figure 121 B shows the combination to described peptide with control rats polyclonal antibody and the mouse IgG HRP of goat Chinese People's Anti-Japanese Military and Political College secondary antibody.Figure 121 C shows the result to the combination of described peptide with anti-B7H3 rat monoclonal antibody and goat anti-rabbit igg HRP secondary antibody.

Figure 112 A-B shows and uses the screening of Phage-ELISA to the output phage from elutriation (panning).For the anti-human CD9 mouse monoclonal antibody of target, phage library is carried out to elutriation.With described target antibody (Figure 122 A) or anti-mouse IgG control antibodies (Figure 123 B), the output phage from the three-wheel elutriation is screened.

Detailed Description Of The Invention

Herein disclosed is for the characterising biological sample (as, from cell culture, organism or experimenter's sample) the method and system of phenotype.Can characterize described phenotype by assessing one or more biomarkers.Described biomarker can be associated with vesica or vesica colony, its vesica surface antigen or vesica useful load for existing.As used herein, the vesica useful load comprises the entity be packaged in vesica.The biomarker that vesica is relevant can comprise membrane-bound and soluble biomarker.Described biomarker can also be the circulating biological mark, such as the microRNA of assessing in body fluid or protein.Unless otherwise specified, mention vesica or biomarker component herein and the term " purifying " that uses or " separation " refer to these components from cell or organism part or purification and separation completely.In addition, unless otherwise specified, vesica that alleged use wedding agent carries out separates and comprises vesica is combined with described wedding agent, and no matter whether this combination makes separating fully of described vesica and other biological entities in parent material.

The method that characterizes phenotype by analysis cycle biomarker (as the biological nucleic acid mark) is described in the scheme 6100A of Figure 61 A, and it is the non-limitative illustration example.Obtain biological sample in first step 6101, as body fluid, tissue sample or cell culture.From described sample, isolating nucleic acid 6103.Described nucleic acid can be DNA or RNA, as microRNA.The biological marking of this phenotype can be provided the assessment of these nucleic acid.Sample by the nucleic acid to relevant to target phenotype (as before disease contrast health, treatment and after treatment), can measure one or more nucleic acid marks as the indication of phenotype.All respects of the present invention relate to by being evaluated at one or more nucleic acid molecule (as microRNA) that exist in described sample determines the biological marking 6105, and the wherein said biological marking is corresponding to predetermined phenotype 6107.Figure 61 B describes the scheme 6100B that uses vesica to separate described nucleic acid molecule in detail.In one embodiment, obtained biological sample 6102, and separated one or more vesicas 6104 from described sample, as the vesica from the specific cells source and/or the vesica relevant to particular disease states.Analyze described vesica 6106 by the existence or the level that characterize the surface antigen associated with described vesica and/or measure the component (" useful load ") existed in described vesica.Unless otherwise specified, term as used herein " antigen " is commonly referred to as biomarker that can combined dose of combination, no matter described wedding agent is antibody, fit, lectin or for other wedding agent of described biomarker, and no matter whether these biomarkers cause immunne response in the host.The vesica useful load can be protein (comprising peptide and polypeptide) and/or nucleic acid (as DNA and RNA).The RNA useful load comprises messenger RNA(mRNA) (mRNA) and microRNA (also being called miRNA or miR herein).Characterize phenotype 6108 according to the biological marking of described vesica.In another illustrative method of the present invention, scheme 6100A implements to characterize phenotype together with 6100B.In such scheme, assessed vesica and nucleic acid (as microRNA), thereby characterized described phenotype.

At a related aspect, this paper provides for finding the method for biomarker, and it comprises vesica surface marker or the useful load mark in sample of assessment and described mark and another sample are compared.The mark of distinguishing between described sample can be used as biomarker of the present invention.These samples can be from experimenter or subject group.For example, described group can be, for example, and for known response person and the non-reactor of the given treatment of given disease or imbalance.Find that the biomarker of distinguishing described known response person and non-reactor provides biological marking treatment (such as therapeutical agent, as medicine or biological products) responded about experimenter's possibility.

Phenotype

Herein disclosed is the product and the method that characterize individual phenotype by analyzing vesica (such as membrane vesicle).Phenotype can be experimenter's any observable feature or proterties, for example prognosis of susceptibility, disease stage or the situation of disease or situation, disease stage or situation stage, disease or situation, physiological status or the reaction to treating.Phenotype can be caused by experimenter's genetic expression and the impact of environmental factors and the interaction between these two, and be caused by the outer genetic modification to nucleotide sequence.

Phenotype in the experimenter can characterize by one or more vesicas that obtain biological sample from the experimenter and analyze described sample.For example, characterize experimenter or individual phenotype and can comprise detection disease or situation (comprising that the commitment before symptom detects), determine prognosis, diagnosis or the treatment diagnosis of disease or situation, or the stage of definite disease or situation or process.Characterizing phenotype can also comprise prediction and the probability analysis, particularly palindromia identified for the suitable treatment in specified disease, situation, disease stage or situation stage or treatment effect, progression of disease, shift diffusion or disease progression.Phenotype can also be type or the hypotype of uniqueness clinically of situation or disease (for example cancer or tumour).Determining of phenotype can also be the determining of physiological situation, organ desperate situation or organ rejection's's (for example, after transplanting) assessment.Product as herein described and method can be assessed the experimenter on individual basis, and it can be provided in the benefit of the more effective and more economical decision-making in treatment.

In one aspect, the present invention relates to the biological marking that vesica analysis provides prediction experimenter possibility to respond to the treatment of disease or imbalance.Characterize phenotype and comprise the respondent that predicts described experimenter/non-respondent's state, wherein the respondent responds to the treatment of disease but not the respondent is reactionless to described treatment.Can in described experimenter, analyze vesica and with known, treatment be responded or responseless previous experimenter's vesica analysis compares.If the biological marking of the vesica in described experimenter and the known previous experimenter that described treatment is responded are more approaching, described experimenter can characterize or be predicted as the respondent of described treatment.Similarly, if the biological marking and known more approaching to the unresponsive experimenter before this of described treatment of the vesica in described experimenter, described experimenter can characterize or be predicted as the non-respondent of described treatment.Described treatment can be for any suitable disease, imbalance or other situation.In the situation that the biological marking of the vesica relevant to respondent/non-respondent's state is known, described method can be used in any disease situation.

Term used in the present invention " phenotype " can refer to contribute to any proterties or the feature of the biological marking of vesica, and this biology marking is used method of the present invention and identifies.For example, phenotype can be the sign that the experimenter may respond to treatment, or more broadly its diagnosis, prognosis or treatment diagnosis that can be the biological marking of the sample survey for the treatment of diagnosis based on for available from the experimenter is determined.

In some embodiments, described phenotype comprises that disease or situation are as listed those in table 1.For example, described phenotype can comprise the existence of tumour, vegetation or cancer or the possibility of Cancer, vegetation or cancer.The cancer that detects or assess by product as herein described or method includes but not limited to mammary cancer, ovarian cancer, lung cancer, colorectal carcinoma, hyperplastic polyp, adenoma, colorectal carcinoma, high grade dysplasia (high grade dysplasia), low dysplasia (low grade dysplasia), hyperplasia of prostate, prostate cancer, melanoma, carcinoma of the pancreas, the cancer of the brain (for example glioblastoma), hematologic malignancies, hepatocellular carcinoma, cervical cancer, carcinoma of endometrium, head and neck cancer, the esophageal carcinoma, gastrointestinal stromal tumor (GIST), renal cell carcinoma (RCC) or cancer of the stomach.Described colorectal carcinoma can be CRC Dukes B or CRC Dukes C-D.Described hematologic malignancies can be B cell lymphocytic leukemia, B cell lymphoma-DLBCL, B cell lymphoma-DLBCL-germinal center sample, B cell lymphoma-DLBCL-activating B cell sample and burkitt's lymphoma.

Described phenotype can be the front situation that cancerates, such as actinic keratosis, atrophic gastritis, leukodermia, erythroplasia, lymphomatoid granulomatosis, the preleukemia, cystic fibrosis, cervical atypical hyperplasia, cervical atypism hyperplasia, xeroderma pitmentosum, Barrett esophagus, colorectal polyp or likely develop into other abnormal structure's growth or focus of malignant tumour.Transformant virus infection such as HIV and HPV also provides the phenotype that can be assessed according to the present invention.

The cancer characterized by method of the present invention can include but not limited to, cancer knurl, sarcoma, lymphoma or leukemia, gonioma, blastoma or other cancer.The cancer knurl includes but not limited to, epithelial tumor, the squamous cell tumor squamous cell carcinoma, the basal cell neoplasms rodent cancer, transitional cell papilloma and cancer, adenoma and gland cancer (body of gland), adenoma, gland cancer, leather bag stomach nesidioblastoma (linitis plastica insulinoma), glucagonoma, gastrinoma, vasoactive intestinal peptide tumor, cholangiocarcinoma, hepatocellular carcinoma, adenocystic carcinoma, the carcinoid of appendix knurl, prolactinoma, pyknocytoma, permitted special Lay Schwann Cells adenoma (hurthle cell adenoma), renal cell carcinoma, grawitz's tumor (grawitz tumor), multiple endocrine adenomas, the uterine endometrium adenoma, annex and appendages of skin tumour, mucoepidermoid tumor, capsule, mucus and serous tumor, cystadenoma, pseudomyxoma peritonei, conduit, leaflet and medullary substance tumour, acinic cell tumor, the combined epithelial tumour, fertile hot tumour (warthin ' s tumor), thymoma, specialization sexual gland tumour, the sex cords mesenchymoma, thecoma, granulosa cell tumor, arrhenoblastoma, the sustenticular cell leydig cell tumor of testis, glomus tumor, chromaffinoma, pheochromocytoma, angioneuromyoma, mole and melanoma, melanocytic nevus, malignant melanoma, melanoma, NM, dysplastic nevus, pernicious mole property melanoma, superficial spreading melanoma and pernicious acra lentigo melanoma.Sarcoma includes but not limited to, the Askin tumour, botryoid sarcoma (botryodies), chondrosarcoma, ewing's sarcoma, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, soft tissue sarcoma, it comprises: alveolar soft part sarcoma, angiosarcoma, cystosarcoma phyllodes, dermatofibrosarcoma, fibroma durum, short desmoplastic small round cell knurl, epithelioid sarcoma, the outer chondrosarcoma of bone, the outer osteosarcoma of bone, fibrosarcoma, hemangiopericytoma, angiosarcoma, Kaposis sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, lymphosarcoma, malignant fibrous histiocytoma, neurofibrosarcoma, rhabdosarcoma and synovial sarcoma.Lymphoma and leukemia include but not limited to, lymphocytic leukemia/small lymphocytic lymphoma, B cell prolymphocytic leukemia, lymphoma lymphoplasmacytic (lymphoplasmacytic lymphoma) (such as

macroglobulinemia), the splenic marginal zone lymphoma, plasma cell myeloma, plasmoma, the monoclonal immunoglobulin storage disorders, heavy chain disease, also be called the lymphadenomatous extranodal marginal zone B cell lymphoma of malt, nodositas marginarium B cell lymphoma (nmzl), follicular lymphoma, lymphoma mantle cell, diffuse large B cell lymphoma, mediastinum (thymus gland) large B cell lymphoid tumor, intravascular large B cell lymphoma, lymphoma primary effusion, burkitt's lymphoma/leukemia, T cell prolymphocytic leukemia, T cell large granular lymphocyte leukemia, aggressive NK chronic myeloid leukemia, adult T-cell leukemia/lymphoma, lymphoma extranodal NK/Tcell, the nose type, enteropathy-type T cell lymphoma, liver splenic t-cell lymphoma, parent cell NK cell lymphoma, mycosis fungoides/Sai Zeli syndrome (sezary syndrome), primary cutaneous CD30 positive T cell lymphoproliferative disease, lymphoma primary cutaneous anaplastic large cell, lymphomatoid papulosis, angioimmunoblastic T cell lymphoma, lymphoma peripheral T cell, non-designated, primary cutaneous type, hodgkin lymphoma classical type (epiloia, cell mixing, rich lymphocyte, lymphocyte exhausts or non-exhausting) and Nodular lymphocyte be the principal mode Hodgkin lymphoma.Germinoma includes but not limited to, gonioma, dysgerminoma, spermocytoma, non-gonioma sexual reproductive cell knurl, embryonal carcinoma, endodermal sinus tumor, choriocarcinoma, teratoma, polyembryoma and gonadoblastoma.Blastoma includes but not limited to, the nephroblastoma, medulloblastoma and retinoblastoma.Other cancer includes but not limited to, lip cancer, laryngocarcinoma, the pharynx cancer, tongue cancer, salivary-gland carcinoma, cancer of the stomach, gland cancer, thyroid carcinoma (oblongata and papillary thyroid carcinoma), kidney, carcinoma of renal parenchyma, cervical cancer, carcinoma of uterine body, carcinoma of endometrium, choriocarcinoma, carcinoma of testis, the urinary system cancer, melanoma, cerebral tumor is (as glioblastoma multiforme, astrocytoma, meningioma, medulloblastoma and peripheral neuroectodermal tumor), carcinoma of gallbladder, bronchogenic carcinoma, multiple myeloma, rodent cancer, teratoma, retinoblastoma, melanoma of choroid, spermocytoma, rhabdosarcoma, craniopharyngioma (craniopharyngeoma), osteosarcoma, chondrosarcoma, myosarcoma, liposarcoma, fibrosarcoma, ewing's sarcoma and plasmoma.

In further embodiment, the cancer of being analyzed can be lung cancer, it comprises that nonsmall-cell lung cancer and small cell lung cancer (comprise small cell carcinoma (oat-cell carcinoma), minicell/maxicell mixed carcinoma and plyability small cell carcinoma), colorectal carcinoma, mammary cancer, prostate cancer, liver cancer, carcinoma of the pancreas, the cancer of the brain, kidney, ovarian cancer, cancer of the stomach, skin carcinoma, osteocarcinoma, the cancer of stomach, mammary cancer, carcinoma of the pancreas, glioma, glioblastoma multiforme, hepatocellular carcinoma, the papillary carcinoma kidney, squamous cell carcinoma of the head and neck, leukemia, lymphoma, myelomatosis or noumenal tumour.

In embodiments, described cancer comprises acute lymphoblastic leukemia; Acute myelogenous leukemia; Adrenocortical carcinoma; The AIDS associated cancer; The AIDS associated lymphoma; Anus cancer; The appendix cancer; Astrocytoma; Atypia teratoblastoma/rhabdoid tumor; Rodent cancer; Bladder cancer; Brain stem glioma; Cerebral tumor (comprising primitive neuroectodermal tumor and pineocytoma on the pineal gland parenchymal tumor, curtain of brain stem glioma, central nervous system atypia teratoblastoma/rhabdoid tumor, central nervous system embryo's sample knurl, astrocytoma, craniopharyngioma, one-tenth ependymoblastoma, ependymoma, medulloblastoma, medulloepithelioma, moderate differentiation); Mammary cancer; Tumor of bronchus; Burkitt's lymphoma; Carcinoma of unknown primary site; Carcinoid tumor; Original site is failed to understand tumour; Central nervous system atypia teratoblastoma/rhabdoid tumor; Central nervous system Embryo tumour; Cervical cancer; The Childhood cancer; Chordoma; Lymphocytic leukemia; Chronic lymphocytic leukemia; Chronic bone marrow proliferation disorder; Colorectal carcinoma; Colorectal carcinoma; Craniopharyngioma; Cutaneous T cell lymphoma; The internal secretion islet cell tumor; Carcinoma of endometrium; Become ependymoblastoma; Ependymoma; The esophageal carcinoma; Esthesioneuroblastema (esthesioneuroblastoma); Ewing's sarcoma; The extracranial germ cell knurl; Extragonadal germ cell tumor; Cholangiocarcinoma; Carcinoma of gallbladder; (stomach) cancer of stomach; The gastrointestinal associated cancers tumour; Patients with gastrointestinal stromal tumors; Gastrointestinal stromal tumor (GIST); Gestational trophoblastic tumor; Neurospongioma; Hairy cell leukemia; Head and neck cancer; The heart cancer; Hodgkin lymphoma; Hypopharyngeal carcinoma; The intraocular melanoma; Islet cell tumor; Kaposis sarcoma; Kidney; Langerhans cell histiocytosis; Laryngocarcinoma; Lip cancer; Liver cancer; Malignant fibrous histiocytoma osteocarcinoma; Medulloblastoma; Medulloepithelioma; Melanoma; The Merkel cell carcinoma; Merkel cell skin carcinoma; Mesothelioma; Hide idiopathic transitivity squamous neck cancer; Oral carcinoma; Multiple endocrine neoplasia syndrome; Multiple myeloma; Multiple myeloma/plasma cell tumor; Cutaneous T cell lymphoma; Myelodysplastic syndrome; The myeloproliferative tumour; CARCINOMA OF THE NASAL CAVITY; Nasopharyngeal carcinoma; Neuroblastoma; Non-Hodgkin lymphoma; Nonmelanoma skin cancer; Nonsmall-cell lung cancer; The mouth cancer; Oral carcinoma; The oropharynx cancer; Osteosarcoma; Other brain and tumor of spinal cord; Ovarian cancer; Epithelial ovarian cancer; Ovarian germ cell tumors; Ovary hangs down pernicious potential tumor; Carcinoma of the pancreas; Papillomatosis; The paranasal sinus cancer; Parathyroid carcinoma; Pelvic cancer; Penile cancer; The pharynx cancer; The pineal gland parenchymal tumor of moderate differentiation; Pineocytoma; Pituitary tumor; Plasma cell tumor/multiple myeloma; The pleura pulmonary blastoma; Primary central nervous system (CNS) lymphoma; Primary hepatocyte hepatocarcinoma; Prostate cancer; The rectum cancer; Kidney; Nephrocyte (kidney) cancer; Renal cell carcinoma; Respiratory cancer; Retinoblastoma; Rhabdosarcoma; The sialisterium cancer; S é zary syndrome; Small cell lung cancer; Carcinoma of small intestine; Soft tissue sarcoma; Squamous cell carcinoma; Squamous neck cancer; Stomach (stomach) cancer; Primitive neuroectodermal tumor on curtain; T cell lymphoma; Carcinoma of testis; Laryngocarcinoma; Thymic carcinoma; Thymoma; Thyroid carcinoma; Transitional cell carcinoma; Renal plevis and ureteral transitional cell carcinoma; Trophoblastic tumor; Carcinoma of ureter; Urethral carcinoma; Uterus carcinoma; Sarcoma of uterus; Carcinoma of vagina; Carcinoma vulvae;

macroglobulinemia or the nephroblastoma.Method of the present invention can be used for characterizing these and other cancer.Therefore, diagnosis, prognosis and the treatment diagnosis of one of cancer disclosed herein can be provided the sign of phenotype.

Described phenotype can also be inflammatory diseases, Immunological diseases or autoimmune disease.For example, described disease can be inflammatory bowel (IBD), Crohn disease (CD), ulcerative colitis (UC), pelvic inflammation, vasculitis, psoriatic, diabetes, autoimmune hepatitis, multiple sclerosis, myasthenia gravis, type i diabetes, rheumatoid arthritis, psoriasis, systemic lupus erythematous (SLE), Hashimoto thyroiditis, Graves disease, ankylosing spondylitis, Sjogrens disease, CREST syndrome, scleroderma, rheumatosis, organ rejection response, primary sclerosing cholangitis or Sepsis.

Described phenotype can also comprise cardiovascular disorder, for example atherosclerosis, congestive heart failure, vulnerable plaque, apoplexy or local asphyxia.Described cardiovascular disorder or situation can be hypertension, stenosis, angiemphraxis or thrombosis event.

Described phenotype can also comprise sacred disease, multiple sclerosis (MS) for example, Parkinson's disease (PD), alzheimer's disease (AD), schizophrenia, bipolar disorder, dysthymia disorders, autism, prion disease, Pick's disease, dull-witted, Huntington Chorea (HD), mongolism, cerebrovascular disease, the Rasmussen encephalitis, viral meningitis, neuropsychiatric systemic lupus erythematous (NPSLE), amyotrophic lateral sclerosis, creutzfeldt-Jacob disease, the Gerstmann-Straussler-Scheinker disease, Transmissible spongiform encephalopathy, ischemical reperfusion injury (for example apoplexy), cerebral trauma, infected by microbes or chronic fatigue syndrome.Described phenotype can also be the situation such as fibromyalgia, chronic neuropathic pain model or peripheral nerve pathologic pain.

Described phenotype can also comprise infectious diseases, for example bacterium, virus or yeast infection.For example, described disease or situation can be Whipples disease, prion disease, liver cirrhosis, methicillin-resistant staphylococcus aureus, HIV, hepatitis, syphilis, meningitis, malaria, tuberculosis or influenza.Can assess virus protein (for example HIV or HCV sample particle) in vesica thus characterize virus status.

Described phenotype can also comprise perinatal period or relevant situation (for example aura eclampsia or premature labor), metabolic trouble or the situation (for example metabolic trouble relevant with iron metabolism or situation) of gestation.The iron that for example, can be determined in vesica is adjusted element (hepcidin) thereby the sign sideropenia.Described metabolic trouble or situation can also for diabetes, inflammation or perinatal period situation.

Method of the present invention can be used for characterizing these diseases or imbalance and the Other diseases that can assess by biomarker or imbalance.Therefore, can provide diagnosis, prognosis and the treatment diagnosis to one of disease disclosed herein or imbalance to the sign of phenotype.

The experimenter

One or more vesicas (as multiple vesica) the biological sample that can obtain from the experimenter by analysis are determined one or more phenotypes of experimenter.Experimenter or patient can include but not limited to Mammals, for example ox, bird, dog, horse, cat, sheep, pig or primate (comprising people and inhuman primate).The experimenter can also comprise: for example, due to the Mammals that becomes important in imminent danger, siberia tiger; The animal farm breeds animal of human consumption (for example for) that perhaps there is economic implications, or the animal (for example as pet or at the zoo domesticated animal) that the mankind is there is to social effect.The example of this type of animal includes but not limited to: zoophagous animal, for example cat and dog; Pig, comprise and raise pig, porker and wild boar; Ruminating animal or ungulate, for example ox, bull, sheep, giraffe, deer, goat, wild ox, camel or horse.In addition, also comprise in imminent danger or at the zoo in the bird raised; And bird, more particularly domestic bird, that is, and poultry, for example turkey and chicken, duck, goose, guinea fowl.Also comprise domestic pig and horse (comprising horse racing).In addition, the any animal species relevant with business activity also all is included, for example, with agricultural, water industry and other movable relevant animal, in described industry, for the safety of economic productivity and/or food chain, the selection of disease surveillance, diagnosis and therapy is conventional measure.

Described experimenter can suffer from disease or the situation existed before this, for example cancer.Perhaps, described experimenter can not suffer from any known situation existed before this.Described experimenter can also be reactive, for example reactive to the treatment right and wrong of cancer to treatment the right and wrong existing or past.

Sample

The biological sample that derives from described experimenter can be any body fluid.For example, described biological sample can be peripheral blood, serum, blood plasma, ascites, urine, cerebrospinal fluid (CSF), phlegm, saliva, marrow, synovia, aqueous humor, amniotic fluid, earwax, milk, bronchoalveolar lavage fluid, seminal fluid (comprising prostatic fluid), examine amber liquid or the liquid of ejaculating in advance, the women penetrates liquid, sweat, movement, hair, tear, capsule liquid, hydrothorax and ascites fluid, pericardial fluid, lymph liquid, the food gruel, chyle, bile, interstitial fluid, menses, fester, sebum, vomitus, vaginal secretions, mucous membrane secretory product, just rare, pancreatic juice, nasal lavage fluid, broncho-pulmonary aspirated liquid or other irrigating solution.Biological sample can also comprise segmentation cavity, Cord blood or parent circulating (its can for fetal origin or maternal source).Described biological sample can also be tissue sample or biological tissue, therefrom can obtain vesica and other circulating biological mark.For example, can cultivate the cell that derives from sample, and separate vesica (for example, referring to embodiment 1) from described culture.In each embodiment, can by these biological samples directly assess biomarker disclosed by the invention or the more particularly biological marking (as, to the existence of nucleic acid or polypeptide biomarker or its functional fragment or the evaluation of level), it makes in all sorts of ways, such as, from blood, blood plasma, extract nucleic acid molecule in serum or any aforementioned biological sample, use protein or antibody array to identify polypeptide (or functional fragment) biomarker, and other array that becomes known for testing and appraisal nucleic acid and peptide molecule in this area, order-checking, PCR and protein technique.

Table 1 has been listed the illustrative example of disease, situation or biological condition, and the respective list of the biological sample that can be analyzed vesica wherein.

Table 1: for various diseases, situation or biological condition, the example of the biological sample of analyzing for vesica

Method of the present invention can be used for using blood sample or blood derivatives to characterize phenotype.Blood derivatives comprises blood plasma and serum.Blood plasma is the liquid ingredient of whole blood, and accounts for 55% of total blood volume.Its mainly a small amount of mineral substance, salt, ion, nutritive substance and the protein in water and solution form.In whole blood, red corpuscle, white corpuscle and platelet suspension are in blood plasma.Serum refers to the blood plasma (that is, whole blood deducts cell and thrombin) of fibrinogen not or other thrombin.

Described biological sample can obtain by the third party, such as a side of the analysis of not carrying out described biomarker, no matter is to the direct assessment of biological sample or carries out somatotype by one or more vesicas to deriving from described biological sample.For example, the experimenter's that described sample can be originated by sample clinician, doctor or other health care management personnel obtain.Perhaps, described biological sample can be obtained by the same side who analyzes vesica.In addition, the biological sample of being analyzed under aseptic condition, file (as, freezing) or store.

The volume of the biological sample of analyzing for biomarker can be 0.1-20mL, for example, lower than approximately 20,15,10,9,8,7,6,5,4,3,2,1 or 0.1mL.

Humoral sample can be used as the sample that characterizes phenotype.For example, can be assessed to provide to the biomarker in sample diagnosis, prognosis and/or the treatment diagnosis of disease.Described biomarker can be the circulating biological mark, such as circulating protein matter or nucleic acid.Described biomarker also can with vesica or vesica cluster correlation.Method of the present invention can be applicable to assess one or more vesicas, and one or more can be present in the different vesica colony in biological sample or experimenter.In biological sample, the analysis of one or more biomarkers can be used for determining whether to obtain other biological sample to be analyzed.For example, to the analysis of one or more vesicas in humoral sample, can contribute to determine whether to obtain the biopsy sample.

Sample from the patient can be gathered under maintenance circulating biological mark and its other target entity condition with the analysis for subsequently comprised.In one embodiment, use CellSave Preservative Tubes (CellSave Preservative Tube) (Veridex, North Raritan, NJ), PAXgene Blood DNA Tubes (Blood DNA Tubes) (QIAGEN GmbH, Germany) one or more and in RNAlater (QIAGEN GmbH, Germany) are processed described sample.

CellSave Preservative Tubes (CellSave pipe) is aseptic vacuum test tube.Each pipe comprises solution, and described solution comprises Na2EDTA and cell preservatives.EDTA absorbs calcium ion, can reduce or eliminate coagulation of blood thus.Described preservatives keeps the form of epithelial cell and other cell and cell-surface antigens to express.Described acquisition and processing is implemented in description in the scheme that can provide according to manufacturers.Each pipe is through emptying to extract the vein whole blood, and it carries out according to standard venesection program known to those of skill in the art.The CellSave pipe is disclosed in U.S. Patent No. 5,466,574; 5,512,332; 5,597,531; 5,698,271; 5,985,153; 5,993,665; 6,120,856; 6,136,182; 6,365,362; 6,551,843; 6,620,627; 6,623,982; 6,645,731; 6,660,159; 6,790,366; 6,861,259; 6,890,426; 7,011,794; 7,282,350; 7,332,288; In 5,849,517 and 5,459,073, it is incorporated to this paper in full with way of reference separately.

Described PAXgene Blood DNA pipe (PAXgene pipe) is for gathering the plastic vacuum pipe of the whole blood that separate nucleic acid uses.The transportation that this pipe can be used for blood collection, whole blood sample and storage and to the separating of the nucleic acid that wherein comprised, as DNA or RNA.Blood enters in the valve tube that comprises additive with the venesection scheme collection of standard.Described acquisition and processing is implemented in description in the scheme that can provide according to manufacturers.The PAXgene pipe is disclosed in U.S. Patent application No.5,906,744; 4,741,446; In 4,991,4,991,104, it all is incorporated to this paper with way of reference separately in full.

Described RNAlater RNA Stabilization Reagent (RNAlater) carries out direct stabilization for the RNA to tissue.RNA may be unsettled in the sample of results.Water-based RNAlater reagent infiltration tissue and other sample, the RNA that stablizes and protect it to comprise thus.This protection contributes to guarantee that downstream analysis is reflected in the rna expression spectrum in this tissue or other sample.After collection, immediately described sample is immersed in the RNAlater reagent of proper volume.Described acquisition and processing is implemented in description in the scheme that can provide according to manufacturers.According to manufacturers, described reagent keeps RNA to reach 1 day most under 37 ℃, 18-25 ℃ lower 7 days or under 2-8 ℃ 4 weeks, thereby allow without liquid nitrogen or dry ice, sample is processed, transports, stored and transports.Described sample also can be placed under-20 ℃ or-80 ℃, the storage of for example filing.The sample of preserving can be used for analyzing the RNA of any type, includes but not limited to total RNA, mRNA and microRNA.RNAlater also can be used for gathering the sample that can be used for DNA, RNA and protein analysis.RNAlater is disclosed in U.S. Patent application No.5, in 346,994, its each be incorporated in full this paper with way of reference.

Vesica

Method of the present invention can comprise one or more vesicas of assessment, and it comprises assessment vesica colony.The vesica that this paper is used is the membrane vesicle come off from cell.Vesica or membrane vesicle include but not limited to: circulation microcapsule bubble (cMV), microcapsule bubble, exosome, nano vesicle, dexosome, bubble, bubble, Prostasomes, particulate, the tube chamber intracellular vesicle, membrane-bound fragment, endosome vesica in tube chamber, endosome sample vesica, the exocytosis medium, the endosome vesica, the vesica of endosome, apoptotic body, multivesicular body, the secretion vesica, phospholipid capsule bubble, liposome vesicle, argosome, texasome, secresome, tolerosome, melanosome, the medium of oncosome or exocytosis.In addition, although vesica can produce by different cell processes, method of the present invention is not limited to or depends on any single mechanism, as long as these vesicas are present in biological sample and can characterize by method disclosed by the invention.Unless otherwise specified, otherwise use the method for a certain vesica to can be used for the vesica of other type.Vesica comprises ball-like structure, and it has the double-layer of lipoid that is similar to cytolemma around inner chamber, and described inner chamber is called as the soluble component of useful load in the time of can including.In some embodiments, method of the present invention has been used exosome, its little secretion vesica that is diameter 40-100nm.For the summary of membrane vesicle (comprising type and character) referring to Thery etc., Nat Rev Immunol.2009 August; 9 (8): 581-93.The properties of dissimilar vesica comprises the character in table 2:

Table 2: vesica characteristic PCR

Abbreviation: phosphatidylserine (PPS); Electron microscopy (EM)

Vesica comprises that the film come off is in conjunction with particle, or claims " particulate ", and it stems from plasma membrane or inner membrance.Vesica can discharge into extracellular environment from cell.The cell that discharges vesica includes but not limited to be derived from or derived from ectoderm, entoderm or mesoblastic cell.Heredity, environment and/or other variation or variation arbitrarily can occur in described cell.For example, described cell can be tumour cell.Vesica can reflect any variation of derived cell, and reflects therefrom the variation in cells of origin, as, there is the cell that range gene suddenlys change.In a kind of mechanism, when the fragment of cytolemma is spontaneously caved in and, finally by exocytosis out the time, vesica generates (such as referring to Keller etc., Immunol.Lett.107 (2): 102-8 (2006)) in cell.Vesica also comprises the structure of the cell source of bilayer lipid membrane institute combination, it by outstanding turn up (foaming), is separated and the sealing of plasma membrane part is produced, perhaps any cell inner membrance by the membrane bound protein that comprises various tumours source generates in conjunction with sending outside of imitated vesicle structure, wherein said membrane bound protein comprises the surface bonding molecule that is derived from host's circulation, this molecular selectivity ground is in conjunction with tumour source protein matter and be included in the molecule in the vesica chamber, and it includes but not limited to microRNA or intracellular protein that tumour is derivative.Bubble and bubble at Charras etc., Nature Reviews Molecular and Cell Biology, p.730-736 the 9th volume Chapter 11, have further description in (2008).From tumour cell come off enter the circulation or body fluid in vesica can be referred to as " circulating tumor source vesica ".When this vesica is exosome, it can be called as circulating tumor source exosome (CTE).In some cases, vesica can be derived from the specific cells source.CTE, as cell source specificity vesica, generally have one or more unique biomarkers, its allow described CTE or cell source specificity vesica for example with in body fluid, separate and some the time in the specificity mode, separate.For example, use the cell or tissue Specific marker with the identification of cell source.Herein disclosed is the example of these cell or tissue Specific markers and its and can further see that tissue-specific gene is expressed and regulation and control (TiGER) database (Tissue-specific Gene Expression and Regulation Database), it can be available from bioinfo.wilmer.jhu.edu/tiger/; Liu etc. (2008) TiGER:a database for tissue-specific gene expression and regulation.BMC Bioinformatics.9:271; Tissue distribution database (TissueDistributionDB), can be available from genome.dkfz-heidelberg.de/menu/tissue_db/index.html.

Vesica can have the diameter that surpasses about 10nm, 20nm or 30nm.Vesica can have over 40nm, 50nm, 100nm, 200nm, 500nm, 1000nm or surpass the diameter of 10,000nm.Vesica can have the diameter of about 30-1000nm, about 30-800nm, about 30-200nm or about 30-100nm.In some embodiments, described vesica has lower than 10,000nm, 1000nm, 800nm, 500nm, 200nm, 100nm, 50nm, 40nm, 30nm, 20nm or lower than the diameter of 10nm.The term " about " that this paper is used when mentioning numerical value means variation higher or lower than described numerical value 10% and belongs in the scope that specified numerical value has.The typical sizes of various types of vesicas is shown in table 2.Can assess vesica to measure the diameter of single vesica or a plurality of vesicas.For example, can measure the diameter range of vesica colony or the mean diameter of vesica colony.The vesica diameter can be used method as known in the art assessment, as, such imaging technique such as electron microscopy.In one embodiment, use optical particle to detect the diameter that (optical particle detection) measures one or more vesicas.The United States Patent (USP) 7,751,053 that is entitled as " Optical Detection and Analysis of Particles " of for example, authorizing referring on July 6th, 2010; And the United States Patent (USP) 7,399,600 that is entitled as " Optical Detection and Analysis of Particles " of mandate on July 15th, 2010.

In some embodiments, in the situation that there is no separation in advance, the purifying or concentrated directly from described biological sample analysis vesica of biological sample.For example, the vesica amount in sample itself can provide the biological marking, and it provides diagnosis, prognosis or treatment diagnosis to determine.Perhaps, can before analysis, from sample, separate, catch, the vesica in purifying or concentrating sample.As described, the separation that this paper is used, catch or purifying comprise with other component in sample partly separate, the part catch or partial purification.Vesica separates and can use various technology implementation as herein described, as, chromatogram, filtration, centrifugal, flow cytometry, affinity capture (as, capture plane or pearl) and/or use micro-fluidic technologies.

Can by by the vesica feature with reference to compare assess vesica as exosome to provide phenotype to characterize.In some embodiments, assessed the surface antigen on the vesica.Described surface antigen can provide the anatomical origin of vesica and/or the sign of cell, and other phenotype information, as neoplastic state.For example, the surface antigen assess patient sample wherein existed for indication colorectum source and cancer (as, body fluid, such as blood, serum or blood plasma) in the vesica of discovery.Described surface antigen can comprise any informedness biological entities that can detect on vesica film surface, and it includes but not limited to surface protein, lipid, carbohydrate and other membrane component.For example, can mean that to the positive detection of the colon source vesica of expressing tumour antigen described patient suffers from colorectal carcinoma.Accordingly, method of the present invention can be used for any disease or the situation that sign is relevant to anatomy or cell derived, and it is by assessment, for example, available from the disease specific of one or more vesicas of experimenter and cell-specific biomarker, completes.

In another embodiment, to one or more vesica useful loads, assessed to provide phenotype to characterize.The useful load of vesica is included in detectable any informedness biological entities in situation about being encapsulated in described vesica, and it includes but not limited to protein and nucleic acid, as, genomic or cDNA, mRNA or its functional fragment, and microRNA (miR).In addition, method of the present invention relates to and detects vesica surface antigen (carry out or gets rid of vesica useful load) so that the phenotype sign to be provided beyond the vesica useful load.For example, can use for the specific wedding agent of vesica surface antigen (as antibody or fit) and identify vesica, and the vesica that can further assess combination is to identify one or more useful load components that wherein disclose.According to described herein, there is target surface antigens or have the target effective load vesica level can with reference to comparing to characterize phenotype.For example, with reference counterpoint, cancer relevant surfaces antigen or vesica useful load (as Tumor-assaciated mRNA or microRNA) cross expressing in sample can indicate as described in the existence of cancer in sample.According to selection and target sample and the desirable comparison with reference to sample of required target sample, the biomarker of assessing can exist or not exist, and improves or reduces.The unrestricted example of target sample comprises: disease; Treatment/not treatment; Different time points, as in longitudinal research; And the unrestricted example with reference to sample: non-disease; Different time points; And to sensitivity or the resistance of candidate therapeutic.

MicroRNA

Can assess at biological sample or in available from the vesica of this class biological sample various biomarker molecules.MicroRNA comprises by a class biomarker of method assessment of the present invention.The microRNA that also is called in this article miRNA or miR is the short rna chain that is about 21-23 Nucleotide.MiRNA is by genes encoding, and it is transcribed by DNA but does not translate becomes protein and therefore comprise non-coding RNA.MiR is processed as being called the short loop-stem structure of front miRNA (pre-miRNA) by the primary transcript that is called former miRNA (pri-miRNA), and finally is processed into obtained strand miRNA.Pre-miRNA generally is formed in autologous complementary region and goes back to the structure of self.These structures are processed by nuclease DIcer subsequently in animal, or by DCL1, are processed in plant.Ripe miRNA molecule and one or more messenger RNA(mRNA)s (mRNA) molecular moiety ground are complementary and can bring into play the function that regulation protein is translated.MiRNA sequence through identifying can obtain in disclosing available database, such as www.microRNA.org, www.mirbase.org or www.mirz.unibas.ch/cgi/miRNA.cgi.

Usually according to UNC " mir-[numeral] " and give numbering to miRNA.The numbering of miRNA is set according to its order of discovery with respect to the miRNA kind of identifying before this.For example, if the last miRNA announced is mir-121, next found miRNA will be named as mir-122, etc.When miRNA is found from known miRNA homology from different organisms, its name can provide [organism identifier]-mir-[numeral] the optional organism identifier symbol of form.Identifier comprises for homo sapiens's hsa with for the mmu of mouse.For example, people's homologue of mir-121 can be described as hsa-mir-121, and the mouse homologue can be described as mmu-mir-121.

Ripe microRNA is given prefix " miR " usually, and gene or precursor miRNA are given prefix " mir ".For example, mir-121 is the precursor of miR-121.When the miRNA of difference gene or precursor are formed to identical ripe miRNA, described gene/precursor can be described by the suffix of numbering.For example, mir-121-1 and mir121-2 can refer to be formed to different genes or the precursor of miR-121.Letter suffix is for indicating the mature sequence be closely related.For example, mir-121a and mir-121b can be formed to respectively miRNA miR-121a and the miR-121b be closely related.In situation of the present invention, use prefix mir-herein ^*or miR- ^*any microRNA (miRNA or miR) of censuring is considered to contain precursor and/or ripe kind, unless clearly stated separately.

Sometimes observe two kinds of ripe miRNA sequences and stem from identical precursor.When one of sequence, than another kind more during horn of plenty, " * " suffix can be used for the more uncommon variant of indication.For example, miR-121 should be main product, and miR-121 ^*to be positioned at the more uncommon variant of finding on the relative arm of precursor.Go out main variant if unidentified, can and distinguish described miR for the suffix " 3p " of the variant from 3 ' arm by the suffix " 5p " of the variant for from precursor 5 ' arm.For example, miR-121-5p stems from 5 ' arm of precursor, and miR-121-3p stems from 3 ' arm.In more uncommon situation, 5p and 3p variant are called as respectively justice (" s ") and antisense (" as ") form.For example, miR-121-5p can be described as miR-121-s, and miR-121-3p can be described as miR-121-as.

The generation in time of above-mentioned UNC is developed and is that generality instructs reference but not absolute regulation.For example, the let of miRNA and lin family continue to use these titles to refer to.Mir/miR convention for precursor/mature form is still to instruct principle, and should consider context is to determine which kind of form that refers to.The further details of miR name is found in www.mirbase.org or Ambros etc., A uniform system for microRNA annotation, RNA 9:277-279 (2003).

Mirnas of plant is followed different UNCs, and it is described in Meyers etc., and Plant Cell.2008 20 (12): in 3186-3190.

Relate to a large amount of miRNA in gene regulating, and miRNA is the part of ever-increasing non-coding RNA classification, it is considered to the main level of Gene Handling now.In some cases, miRNA can by embed 3 of its said target mrna '-regulatory site in UTR disturbs translation, thereby caused checking of translation.Target identification relates to the complementary base pairing of target site and the seed region of stating miRNA (the 2-8 position of described miRNA 5 ' end), although plant that complementary levels of precision is not measured exactly and can be modified by 3 ' pairing.In other cases, miRNA and siRNA (siRNA) are brought into play function similarly, and the mRNA sequence that is incorporated into complete complementary is to destroy the target transcript.

The sign of multiple miRNA shows that they affect various procedures, comprise early development, cell proliferation and necrocytosis, apoptosis and metabolism of fat.For example, shown that some miRNA (such as lin-4, let-7, mir-14, mir-23 and bantam) plays a crucial role in cytodifferentiation and tissue development.Other miRNA it is believed that the room and time expression pattern due to they difference has similar important effect.

Can be available from miRBase (www.mirbase.org) but the miRNA database comprise the searching database of delivering miRNA sequence and annotation.Further information about miRBase is found in following article, each article is incorporated to this paper: Griffiths-Jones etc., miRBase:tools for microRNA genomics.NAR 2,008 36 (Database Issue): D154-D158 in full by reference; Griffiths-Jones etc., miRBase:microRNA sequences, targets and gene nomenclature.NAR 2,006 34 (Database Issue): D140-D144; And Griffiths-Jones, S.The microRNA Registry.NAR 2,004 32 (Database Issue): D109-D111.Representative miRNA is contained in the 16th phase distribution (Release 16) of miRBase, and it can obtain from June, 2010.

According to described herein, the known participation cancer of microRNA and Other diseases and can be assessed with for characterizing the phenotype of sample.For example, referring to, Ferracin etc., Micromarkers:miRNAs in cancer diagnosis and prognosis, Exp Rev Mol Diag, in April, 2010, the 10th volume, the 3rd phase, 297-308 page; Fabbri, miRNAs molecular biomarkers of cancer, Exp Rev Mol Diag, in May, 2010, the 10th volume, the 4th phase, 435-444 page.The technology of separation and sign vesica and miR it is known to those skilled in the art that.Except method provided herein, other method is found in the U.S. Patent No. 7 that is entitled as " METHODS FOR ASSESSING RNA PATTERNS " of authorizing on February 15th, 2011, 888, 035, and on November 30th, 2010 submit to be entitled as " METHODS AND SYSTEMS FOR ISOLATING, STORING, AND ANALYZING VESICLES " international patent application No.PCT/US2010/058461 and the PCT/US2011/021160 that is entitled as " DETECTION OF GASTROINTESTINAL DISORDERS " submitted on January 13rd, 2011, each application is incorporated to this paper in full by reference.

The circulating biological mark

The circulating biological mark is included in detectable biomarker in body fluid (as blood, blood plasma, serum).The example of circulation biomarker for cancer comprises the prostate specific antigen (PSA) of serum cardiac troponin T (cTnT), prostate cancer and the CA125 of ovarian cancer.Circulating biological mark of the present invention comprises the arbitrarily suitable biomarker that can detect in body fluid, and it includes but not limited to protein, nucleic acid (as DNA, mRNA and microRNA), lipid, carbohydrate and metabolite.The circulating biological mark can comprise the biomarker do not combined with cell, such as membrane-bound, be embedded in part in membrane-bound fragment, biological composite or be free on the biomarker in solution.In one embodiment, the circulating biological mark is the biomarker relevant to one or more vesicas that exist in experimenter's biological liquid.

Identified for characterizing the circulating biological mark of various phenotypes.For example, referring to Ahmed N, etc., Proteomic-based identification of haptoglobin-1 precursor as a novel circulating biomarker of ovarian cancer.Br.J.Cancer 2004; Mathelin etc., Circulating proteinic biomarkers and breast cancer, the Gynecol Obstet Fertil.2006 7-8 month; 34 (7-8): 638-46.2006 electronic publishing on July 28; Ye etc., Recent technical strategies to identify diagnostic biomarkers for ovarian cancer.Expert Rev Proteomics.2007 February; 4 (1): 121-31; Carney, Circulating oncoproteins HER2/neu, EGFR and CAIX (MN) as novel cancer biomarkers.Expert Rev Mol Diagn.2007 May; 7 (3): 309-19; Gagnon, Discovery and application of protein biomarkers for ovarian cancer, Curr Opin Obstet Gynecol.2008 February; 20 (1): 9-13; Pasterkamp etc., Immune regulatory cells:circulating biomarker factories in cardiovascular disease.Clin Sci (Lond) .2008 August; 115 (4): 129-31; Fabbri, miRNAs molecular biomarkers of cancer, Exp Rev Mol Diag, in May, 2010, Vol.10, No.4,435-444 page; PCT patent publication No. WO/2007/088537; United States Patent (USP) 7,745,150 and 7,655,479; U.S. Patent Publication No. 20110008808,20100330683,20100248290,20100222230,20100203566,20100173788,20090291932,20090239246,20090226937,20090111121,20090004687,20080261258,20080213907,20060003465,20050124071 and 20040096915, each open this paper that is incorporated to by reference in full.

The vesica enrichment

Can be before analyzing and/or during separation, purifying, concentrated or additionally enrichment vesica or vesica colony.Unless otherwise specified, mention vesica or biomarker component herein and the term " purifying " that uses or " separation " comprise these components from the part of cell or organism or purification and separation completely.Can purifying or concentrating vesicles before analyzing.The analysis of vesica can comprise the amount of one or more vesica colonies of quantitative biological sample.For example, can carry out the heterogeneous population of vesica quantitatively, perhaps can be separated by the heterogeneous population of vesica the vesica colony (for example there is particular organisms marker profile, the specific biological marking or be derived from the vesica colony of specific cell type) of homogeneous, and carry out quantitatively.As described herein, the vesica analysis can also comprise quantitatively or detect qualitatively one or more specific biomarkers distribution or biological markings of vesica.

Vesica for example can store and file in biofluid storehouse (bio-fluid bank), and fetches as required with for analyzing.In addition, vesica can also separate and obtain in the biological sample that in the experimenter of live body or death, has gathered or stored before.In addition, vesica can be by according to King etc., and the biological sample of collecting described in Breast Cancer Res 7 (5): 198-204 (2005) separates and obtains.Vesica can be obtained by filing or sample separation that store.Perhaps, vesica can separate and obtains and without storing or filing sample and being analyzed in biological sample.In addition, the third party can obtain or store described biological sample, or obtains or store described vesica with for analyzing.

The vesica colony of enrichment can be obtained by biological sample.For example, can catch with size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanometer film ultrafiltration, immunosorption, affinity purification, microfluidic separation or their combination come concentrated from biological sample or separate vesica.

Can use size exclusion chromatography (example gel infiltration post), centrifugal or density gradient centrifugation and filter method.For example, can pass through differential centrifugation, anionresin and/or gel permeation chromatography (for example, in U.S. Patent No. 6,899,863 and 6,812, described in 023), sucrose density gradient, organoid electrophoresis (for example, in U.S. Patent No. 7,198, described in 923), Magnetic activated cell sorting art (MACS) or nanometer film ultrafiltration and concentration device separate vesica.Can also use the various combinations of separation or concentration method.

High-abundance proteins matter (for example albumin and immunoglobulin (Ig)) may hinder vesica separating from biological sample.For example, can come to separate exosome from biological sample by the system of having utilized Multiple Antibodies, described antibody for example, be specific for existing rich in protein in biological sample (blood).This system can be removed multiple proteins once, shows thus more low-abundance material, for example the specific vesica of cell source.

Such system can be for for example, separating vesica from biological sample (blood, cerebrospinal fluid or urine).In addition, can also pass through at J Proteome Res 2004 such as Chromy; The removal method of the high-abundance proteins matter described in 3:1120-1127 strengthens the separation of vesica in biological sample.In another embodiment, can also be according to Zhang etc., Mol Cell Proteomics 2005; The described use glycopeptide of 4:144-155 is caught the removal serum protein, and strengthens the separation of vesica in biological sample.In addition, can be as Pisitkun etc., Proc Natl Acad Sci U S A, 2004; Described in 101:13368-13373 by differential centrifugation, then with the antibody of epi-position for tenuigenin or anti cytoplasmic, contact the vesica of separation of biological samples (for example urine).

Can also strengthen separation or the enrichment of vesica in biological sample by using sonication (for example, by applying ultrasonic wave) or stain remover, other film activator or their any combination.For example, ultrasonic energy can be applied to potential tumor sites, and be not limited by theory, can increase the release of vesica in tissue, thereby can analyze or assess the vesica colony from the enrichment of biological sample by one or more methods disclosed herein.

Sample preparation

By the method (separation as affine as antibody) of detection circulating biological mark described herein, can use in case of necessity various concentrated or separable programmings to optimize the consistence of acquired results.These steps can comprise stirring (such as vibration or vortex), different isolation technique (such as the separation as PEG based on polymkeric substance) and filter and other step during be concentrated into different levels.Those skilled in the art should understand, and these processing can be carried out containing each different steps of the sample of vesica at test pack.In one embodiment, described sample self (as, body fluid, such as blood plasma or serum) carry out vortex.In some embodiments, after one or more sample preparation steps (separating as, vesica) are carried out, described sample is carried out to vortex.Can be as required at some or all, in suitable sample preparation step, be stirred.Additive be can in each different step, introduce to improve described processing, for example, gathering or the degraded of target organism mark controlled.

Also can be as required by using the described sample of various different agent treated to optimize described result.These reagent comprise additive for controlling gathering or for regulating the additive of pH or ionic strength.Control the additive of assembling and comprise encapsulant (such as bovine serum albumin (BSA) and milk), chaotropic agent (Guanidinium hydrochloride) and stain remover or tensio-active agent.Available ionic detergent comprises sodium lauryl sulphate (SDS, Sodium Lauryl Sulphate BP/USP (SLS)), laureth sodium sulfate (SLS, Zetesol NL (SLES)), Texapon Special (ALS), Cetrimonium Bromide (cetrimonium bromide), cetrimonium chloride (cetrimonium chloride), cetrimonium stearate (cetrimonium stearate) etc.Available nonionic (zwitter-ion) stain remover comprises polyoxyethylene glycol, polysorbate20 (also being called polysorbas20), other polysorbate class (as 40,60,65,80 etc.), Triton-X (as X100, X114), 3-[(3-courage amido propyl) dimethylamino]-1-propanesulfonic acid (CHAPS), CHAPSO, Septochol, Sodium desoxycholate, NP-40, glucosides class, octyl group-glucosinolate, maltoside etc.In some embodiments, Pluronic F-68 (a kind of tensio-active agent that reduces platelet aggregation that shows) be used to separate and/or detection period between pack processing containing the sample of vesica.F68 can be used under 0.1% to 10% concentration, for example, and 1%, 2.5% or 5% concentration.Can use various acid, alkali, buffer reagent or salt to regulate pH and/or the ionic strength of described solution, it includes but not limited to, salt solution (TBS), sodium phosphate, Repone K, potassiumphosphate, Trisodium Citrate and salt solution-Trisodium Citrate (SSC) buffer reagent of sodium-chlor (NaCl), phosphate buffered saline (PBS) (PBS), tris buffering.In some embodiments, add NaCl, for example 1%, 2.5% or 5% ultimate density with 0.1% to 10% concentration.In some embodiments, add polysorbas20, for example 0.05%, 0.25% or 0.5% ultimate density with 0.005% to 2% concentration.Comprise inert protein for encapsulant of the present invention, as milk-protein, degreasing dry milk albumen (non-fat dry milk protein), albumin, BSA, casein or serum, such as new-born calf serum (NBCS), lowlenthal serum, rabbit anteserum or salmon serum.Described protein can 0.1% to 10% concentration add, the concentration as 1%, 2%, 3%, 3.5%, 4%, 5%, 6%, 7%, 8%, 9% or 10%.In some embodiments, the concentration that BSA can 0.1% to 10% adds, the concentration as 1%, 2%, 3%, 3.5%, 4%, 5%, 6%, 7%, 8%, 9% or 10%.In one embodiment, according to the method provided in the U.S. Patent application 11/632946 of submitting on July 13rd, 2005, process described sample, this application is incorporated to this paper in full by reference.Can use commercially available encapsulant, such as SuperBlock, StartingBlock, the Protein-Free from Pierce (Thermo Fisher Scientific, Rockford, the branch of IL).In some embodiments, the concentration with 0.1% to 10% add the SSC/ stain remover (as, there is the 20X SSC of the Triton-X 100 of 0.5% polysorbas20 or 0.1%), for example, with 1.0% or 5.0% concentration.

Can use as required the method for various various combination optimum detection vesicas He other circulating biological mark of scheme as herein described and processing.Can be by the various various combination optimum detection schemes of stirring, separation method and additive.In some embodiments, vortex patient sample before separating step and afterwards, and use encapsulant (comprising BSA and/or F68) to process described sample.These processing can reduce the formation of large aggregation or protein or other bioclastic, and therefore more consistent detected result output is provided.

Strainer

Can pass through to filter the biological sample from the experimenter with filtration module, thereby and collect from described filtration module the retentate that comprises vesica and separate vesica and separate vesica from biological sample from described biological sample.Described method can comprise, filters the biological sample from the experimenter by the filtration module that comprises strainer, and collects from described filtration module the retentate that comprises vesica, separates vesica from described biological sample therefrom.In one embodiment, described filter is held back and is surpassed the approximately molecule of 100 kilodaltons.

Described method can further comprise the biological marking of measuring vesica.Described method also can further comprise retentate is applied to multiple substrate, wherein each substrate is coupled to one or more trapping agents, and trapping agent or trapping agent that each subgroup of described multiple substrate comprises another subgroup that is different from described multiple substrate combine.

This paper also provides the method for the biological marking of the vesica in the working sample, it comprises: by filtration module, filter the biological sample from the experimenter who suffers from illness, collect the retentate that comprises one or more vesicas from described filtration module, and measure the biological marking of described one or more vesicas.In one embodiment, described filtration module comprises to hold back and surpasses the approximately strainer of the molecule of 100 or 150 kilodaltons.

Method disclosed herein can further comprise the phenotype that characterizes the experimenter, and it collects by with filtration module, filtering the biological sample from the experimenter retentate that comprises one or more vesicas from described filtration module; Detect the biological marking of described one or more vesicas; And realize according to described biological marking sign experimenter's phenotype, wherein characterize the sensitivity with at least 70%.In some embodiments, characterize the amount of measuring one or more vesicas with described biological marking that comprises.In addition, described sign can have approximately 80% to 100% sensitivity.

This paper also provides the method for the analysis of multiplexing multiple vesica.In some embodiments, described method comprises by filtration module and filters the biological sample from the experimenter; Collect the retentate that comprises described multiple vesica from described filtration module; Described multiple vesica is applied to multiple trapping agent, and wherein said multiple trapping agent is coupled to a plurality of substrates, and another subgroup of each subgroup of described a plurality of substrates and described multiple substrate difference mark differently; Catch at least one subgroup of described multiple vesica; And determine the biological marking at least one subgroup of the described vesica that is hunted down.In one embodiment, each substrate is coupled to one or more trapping agents, and each subgroup of described a plurality of substrates comprises and compares the combination of different trapping agents or trapping agent from another subgroup of described a plurality of substrates.In some embodiments, at least one subgroup intrinsic ground mark of described a plurality of substrates, as comprise one or more marks.Described substrate can be particle or pearl, or its arbitrary combination.In one embodiment, described filtration module comprises strainer, and it is held back and surpasses the approximately molecule of 100 or 150 kilodaltons.

In some embodiments, for the method for the analysis of multiplexing multiple vesica, comprise by filtration module and filter the biological sample from the experimenter, wherein said filtration module comprises to hold back and surpasses the approximately strainer of the molecule of 100 kilodaltons; Collect the retentate that comprises described multiple vesica from described filtration module; Described multiple vesica is applied to multiple trapping agent, and wherein said multiple trapping agent is coupled to microarray; Catch at least one subgroup of described multiple vesica on described microarray; And determine the biological marking at least one subgroup of the described vesica that is hunted down.In one embodiment, described filtration module comprises strainer, and it is held back and surpasses the approximately molecule of 100 or 150 kilodaltons.

Can before being separated by filtration, purify described biological sample.For example, can remove non-vesica component as cell debris.Described purification can be undertaken by low-speed centrifugal, all 5,000 * g according to appointment, 4,000 * g, 3,000 * g, 2,000 * g, 1,000 * g or lower.Subsequently the biological sample of the supernatant liquor that comprises described vesica or purification is collected and filtered with from described decontamination of biological sample separation vesica.In some embodiments, biological sample was not purified before by the filtering separation vesica.

In some embodiments, do not use high speed centrifugation from the sample separation vesica, as ultracentrifugation.For example, separation may not need to use all 100,000 * g according to appointment or higher centrifugal speed.In some embodiments, used the centrifugal speed lower than 50,000 * g, 40,000 * g, 30,000 * g, 20,000 * g, 15,000 * g, 12,000 * g or 10,000 * g from the sample separation vesica.

For from the filtration module of sample separation vesica, being based on the filter core of fiber.For example, described fiber can be the polymer fiber of hollow, as polypropylene hollow fiber.Can by using, such as peristaltic pump, such pumping unit enter described module by sample fluid (such as biofluid disclosed by the invention) pumping, thus biological sample is introduced in described filtration module.Flow rate pump is variable, and all according to appointment 0.25,0.5,1,1.5,2,2.5,3,3.5,4,4.5,5,6,7,8,9 or 10mL/ minute.

Described filtration module can be film filter module.For example, described film filter module can comprise the filter disc film, such as hydrophilic poly(vinylidene fluoride) (PVDF) the filter disc film be assemblied in stir chamber instrument (as comprising magnetic stirring apparatus).In some embodiments, described sample is because the pressure gradient of setting up at described filter membrane either side is passed through strainer.

Described strainer can comprise the material with low hydrophobic adsorptivity and/or high water-wet behavior.For example, described strainer can have mean pore size and the hydrophilic surface that retains vesica and see through most protein, limit protein matter absorption thus.For example, described strainer can comprise and selects free polypropylene, PVDF, polyethylene, fluorinated ethylene propylene, Mierocrystalline cellulose, Cellulose diacetate, polyvinyl alcohol and ethylene-vinyl alcohol (EVAL

kuraray Co., Okayama, Japan) material of the group that forms.Other material can be used in strainer includes but not limited to, polysulfones and polyethersulfone.

Described filtration module can have to hold back and surpasses the approximately strainer of the molecule of 50,60,70,80,90,100,110,120,130,140,150,160,170,180,190,200,250,300,400 or 500 kilodaltons (kDa), such as having the approximately strainer of 50,60,70,80,90,100,110,120,130,140,150,160,170,180,190,200,250,300,400 or 500 MWCO (weight shutoff value).In some embodiments, the filters in filtration module is had an appointment 0.01 μ m to the about mean pore size of 0.15 μ m, and in some embodiments, has approximately 0.05 μ m to the about mean pore size of 0.12 μ m.In some embodiments, described this filter has the approximately mean pore size of 0.06 μ m, 0.07 μ m, 0.08 μ m, 0.09 μ m, 0.1 μ m or 0.11 μ m.

Described filtration module can be commercially available post, such as being generally used for proteins concentrate or for separating of the post of protein.Example includes but not limited to, from the post of Millpore (Billerica, MA), such as Amicon

centrifugal filter, or from Pierce the post of (Rockford, IL), such as Pierce Concentrator filter for installation.From the available post of Pierce comprise there is 9kDa, the disposable ultrafiltration centrifugal device of the MWCO of 20kDa and/or 150kDa.These thickeners are comprised of the high-performance regenerated cellulose film of being combined with the circular cone device.Described strainer can be as United States Patent (USP) 6,269, describes in 957 or 6,357,601, and two applications all are incorporated to this paper in full by reference.

Can collect the retentate that comprises separated vesica from described filtration module.Can collect this retentate by from described strainer, rinsing described retentate.Not bound by theory, the strainer of selecting to have the water-wetted surface characteristic form and thus the absorption of limit protein matter can be used for collecting more simply described retentate and be down to minimum by the use of collection technique harsh or consuming time.

Can further describe as the present invention, subsequently collected retentate is used for to analysis subsequently, such as the biological marking of one or more vesicas in the described retentate of assessment.Can on collected retentate, directly implement described analysis.Perhaps, can before being analyzed, one or more vesicas further concentrate or the collected retentate of purifying.For example, can use that size exclusion chromatography, density gradient centrifugation, differential centrifugation, immunosorption are caught, affinity purification, microfluidic separation or their combination, describe such as the present invention, further concentrate described retentate or further separate vesica from described retentate.In some embodiments, described retentate can carry out another step filtration.Perhaps, using before strainer separates vesica, with size exclusion chromatography, density gradient centrifugation, differential centrifugation, immunosorption catch, affinity purification, microfluidic separation or their combination concentrate or separate described vesica.

For example, surpassing approximately 50 by having to hold back, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, the strainer of the molecule of 400 or 500 kilodaltons (kDa) is (such as having approximately 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, the strainer of 400 or 500 MWCO (weight shutoff value)) before filtration module filtering biological sample, can be at first by thering is approximately 0.01 μ m to about 2 μ m, approximately 0.05 μ m is to approximately the hole of 1.5 μ m or the strainer in aperture filter described biological sample.In some embodiments, the described filters aperture of 0.5,0.6,0.7,0.8,0.9,1.0,1.1,1.2,1.3,1.4,1.5,1.6,1.7,1.8,1.9 or 2.0 μ m of having an appointment.Described strainer can be syringe filter.Therefore, in one embodiment, described method is included in by comprising to hold back and surpasses the approximately filtration module of the strainer of the molecule of 100 or 150 kilodaltons and filter described biological sample by strainer (such as syringe filter) before filtering described sample, and wherein said syringe filter has over the about hole of 1 μ m.In one embodiment, described strainer be the strainer of 1.2 μ M and after filtering by described sample the 7ml by thering is the 150kDa cutoff or the thickener post of 20ml.

Described filtration module can be the parts of microfluidic device.The microfluidic device that can also be called " chip lab " system, biomedical microelectromechanical systems (bioMEM) or multi-part integrated system can be used for separating and analyzing vesica.This type systematic makes treating processes and other process (process further described such as the present invention) microminiaturization and the compartmentation that allows vesica combination, biomarker to detect.

Microfluidic device also can separate for vesica by comprising filtration module.For example, microfluidic device can be used one or more passages to separate from biological sample with the large young pathbreaker's vesica according to biological sample.Biological sample can be introduced to one or more microfluidic channel, it optionally allows vesica to pass through.Described microfluidic device can further comprise wedding agent or surpass a kind of filtration module for example, selects vesica with the characteristic according to described vesica (, size, shape, deformability, biomarker distribute or the biological marking).

Wedding agent

Wedding agent is the reagent in conjunction with circulating biological mark (such as the component of vesica or vesica).Described wedding agent can be used as trapping agent and/or detection agent.Trapping agent can in conjunction with and catch the circulating biological mark, such as, by the component in conjunction with vesica or biomarker, undertaken.For example, described trapping agent can be capture antibody or capture antigen, and it is incorporated into the antigen on vesica.Detection agent can be incorporated into the circulating biological mark, helps thus the detection to described biomarker.For example, comprise the antigen isolated with substrate or fit trapping agent can be used for catching the vesica in sample, and comprise the antigen or the fit detection agent that carry mark and can be used for detecting caught vesica by the detection of the marker to detection agent.In some embodiments, use trapping agent and the detection agent assessment vesica of the identical vesica biomarker of identification.For example, can use four transmembrane proteins to catch vesica colony (such as the anti-CD9 antibody that is incorporated into substrate by use), and the vesica that can use fluorescently-labeled anti-CD9 antibody labeling to catch, thereby the vesica of catching detected.In other embodiments, use trapping agent and the detection agent assessment vesica of the different vesica biomarkers of identification.For example, can use the cell-specific mark to catch vesica colony (such as the anti-PCSA antibody that is incorporated into substrate by use), and the vesica that can use fluorescently-labeled anti-CD9 antibody labeling to catch, thereby the vesica of catching detected.Similarly, can use general vesica mark to catch vesica colony (such as the anti-CD9 antibody that is incorporated into substrate by use), and can use the vesica of catching for the fluorescent-labeled antibody mark of cell-specific or disease specific mark, thereby detect the vesica of catching.

In one embodiment, use the trapping agent that is incorporated into the biomarker on vesica to catch vesica.According to the present invention, further describe, described trapping agent can be coupled to substrate and for separating of vesica.In one embodiment, affinity capture or the separation for the vesica to being present in substrate or sample by trapping agent.

Can after concentrated from biological sample or separation vesica, use wedding agent.For example, before separating or detecting and there is the vesica of the particular organisms marking, can at first from biological sample, separate vesica.Can use the wedding agent separation of described biomarker or detect the vesica with particular organisms marking.Can separate or detect the vesica with particular organisms marking from the heterogeneous population of vesica.Perhaps, can use wedding agent to the biological sample that comprises vesica in the situation that do not carry out prior separation or enrichment step.For example, in connection with agent for directly from biological sample, separating or detect the vesica with particular organisms marking.

Wedding agent can be nucleic acid, protein or can be in conjunction with other molecule of vesica component.Described wedding agent can comprise chemical compound (including but not limited to medicine, labelled reagent), arborescence or their combination of DNA, RNA, monoclonal antibody, polyclonal antibody, Fab, Fab ', single-chain antibody, synthetic antibody, fit (DNA/RNA), class peptide, zDNA, peptide nucleic acid(PNA) (PNA), lock nucleic acid (LNA), lectin, synthetic or natural formation.For example, described wedding agent can be capture antibody.In embodiments of the invention, described wedding agent is the membrane protein labelling agent.For example, referring to, be disclosed in the membrane protein labelling agent in the U.S. Patent Publication No. US 2005/0158708 of Alroy etc.In one embodiment, describe according to the present invention and separate or catch vesica, and by one or more membrane protein labelling agent for detection of described vesica.

In some cases, can separate or detect vesica with single wedding agent.In other situation, can separate or detect vesica with the combination of different wedding agents.For example, can with at least 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,25,50,75 or 100 kind of different wedding agent carry out to separate or detect vesica from biological sample.In addition, according to hereinafter further describing, can form the biological marking of vesica for one or more different wedding agents of vesica.

Can also carry out multiplexing with different wedding agents.For example, thus can separate or detect various vesica colony by the wedding agent with different and implement separation or the detection more than a kind of vesica colony.Different wedding agents can from different particle combinations, wherein said different particle is labeled.In another embodiment, can be by the array that comprises different wedding agents for multiple analysis, wherein said different wedding agent is by the difference ground mark, or can determine the position on array according to wedding agent.Can under the resolving power of protrude mark thing or detection method, complete multiplexing, as described hereinafter.Can be by described wedding agent for detection of vesica, such as for detection of cell source specificity vesica.A kind of wedding agent or multiple wedding agent itself can form wedding agent and distribute, and it provides the biological marking of vesica.One or more wedding agents can be selected from Fig. 2.For example, if use 2 kinds, 3 kinds, 4 kinds or more kinds of wedding agent to detect at the Difference test of the vesica of the heterogeneous assorted colony of vesica or in separating or separate vesica colony, the particular combination agent of described vesica colony distributes provides the biological marking of this specific vesica colony.Can use multiple wedding agent to detect vesica in multiple mode.Therefore, described wedding agent also can be used for forming the biological marking of vesica.The described biological marking can be used for characterizing phenotype.

Described wedding agent can be lectin.Lectin is optionally in conjunction with the protein of polysaccharide and glycoprotein, and is distributed in plant and animal widely.For example, can use lectin from Snow Lotus Herb lectin (" the GNA ") form of snowdrop (Galanthus nivalis) for separating vesica, from the lectin of daffodil lectin (" the NPA ") form of daffodil (Narcissus pseudonarcissus) or from the lectin (Boyd etc. of " blue algae antiviral protein (cyanovirin) " by name of the ellipse spore nostoc of blue green algae (Nostoc ellipsosporum), Antimicrob Agents Chemother 41 (7): 15211530,1997; Hammar etc., Ann N Y Acad Sci 724:166 169,1994; Kaku etc., Arch Biochem Biophys 279 (2): 298 304,1990).These lectins can be incorporated into the glycoprotein with high mannose content, and (Chervenak etc., Biochemistry 34 (16): 5,685 5695,1995).High mannose glycoprotein refers to has α-1 → 3 or the glycoprotein that connects of the seminose of α-1 → 6 seminose-seminose key form-seminose.

Described wedding agent can be the material in conjunction with one or more lectins.Lectin is caught the separation that can be applicable to biomarker cathepsin D, and this is because it is for can binding lectin Snow Lotus Herb lectin (GNA) and the glycosylated protein of concanavalin A (ConA).

Use lectin to be described in the International Patent Application PCT/US2010/058461 that is entitled as " METHODS AND SYSTEMS FOR ISOLATING; STORING, AND ANALYZING VESICLES " submitted on November 30th, 2010 with the method and apparatus of catching vesica; The PCT/US2009/066626 that is entitled as " AFFINITY CAPTURE OF CIRCULATING BIOMARKERS " that on December 3rd, 2009 submits to; The PCT/US2010/037467 that is entitled as " METHODS AND MATERIALS FOR ISOLATING EXOSOMES " that on June 4th, 2010 submits to; And the PCT/US2007/006101 that is entitled as " EXTRACORPOREAL REMOVAL OF MICROVESICULAR PARTICLES " of submission on March 9th, 2007, each application all is incorporated to this paper in full by reference.

Wedding agent can be antibody.For example, can there are with one or more antigens to existing on vesica specific one or more antibody and separate vesica.For example, vesica can have CD63 in its surface, and can be for separating of described vesica for antibody or the capture antibody of CD63.Perhaps, the vesica that is derived from tumour cell can be expressed EpCam, can use the antibody for EpCam and CD63 to separate described vesica.Other antibody for separating of vesica can comprise antibody or the capture antibody for CD9, PSCA, TNFR, CD63, B7H3, MFG-E8, EpCam, Rab, CD81, STEAP, PCSA, PSMA or 5T4.Other antibody for separating of vesica can comprise antibody or the capture antibody for DR3, STEAP, epha2, TMEM211, MFG-E8, tissue factor (TF), unc93A, A33, CD24, NGAL, EpCam, MUC17, TROP2 or TETS.

In some embodiments, described trapping agent is the antibody for CD9, CD63, CD81, PSMA, PCSA, B7H3, EpCam, PSCA, ICAM, STEAP or EGFR.Trapping agent also can be used for identifying the biomarker of vesica.For example, trapping agent is as the biomarker of the antibody recognition CD9 for CD9 as vesica.Can use multiple trapping agent in some embodiments, such as in multiple analysis.Described multiple trapping agent can comprise for one or more the wedding agent in CD9, CD63, CD81, PSMA, PCSA, B7H3, EpCam, PSCA, ICAM, STEAP and EGFR.In some embodiments, described multiple trapping agent comprises the wedding agent for CD9, CD63, CD81, PSMA, PCSA, B7H3, MFG-E8 and/or EpCam.In other embodiment again, described multiple trapping agent comprises the wedding agent for CD9, CD63, CD81, PSMA, PCSA, B7H3, EpCam, PSCA, ICAM, STEAP and/or EpCam.Described multiple trapping agent comprises the wedding agent for TMEM211, MFG-E8, tissue factor (TF) and/or CD24.

The mentioned antibody of this paper can be the immunocompetence part of immunoglobulin molecules or immunoglobulin molecules,, comprises molecule and the synthetic antibody of the antigen binding site of conjugated antigen specifically that is.Immunoglobulin molecules can be any kind (for example IgG, IgE, IgM, IgD or IgA) or the subclass of immunoglobulin molecules.Antibody includes but not limited to polyclonal antibody, monoclonal antibody, bi-specific antibody, synthetic antibody, humanized antibody, chimeric antibody, single-chain antibody, Fab fragment and F (ab ') ₂fragment, Fv or Fv ' partly, the epi-position binding fragment of the fragment that produced by the Fab expression library, antiidiotype (anti-Id) antibody or any antibody mentioned above.If antibody preferentially is combined with antigen, and for example with another kind of molecule, have lower than about 30%, 20%, 10%, 5% or 1% cross reactivity, this antibody, or any molecule generally, " combination specifically " antigen (or other molecule).

Wedding agent can also be polypeptide or peptide.Polypeptide is used with the understanding of broad sense, and can comprise subunit amino acid, amino acid analogue or intend the sequence of peptide.Described subunit can connect by peptide bond.Described polypeptide can be the polypeptide of naturally occurring, the natural form processing (for example passing through enzymatic digestion) that has a polypeptide, chemosynthesis or recombinant expressed.Technology that can Application standard is carried out chemosynthesis to the polypeptide for method of the present invention.Described polypeptide can comprise the amino acid (such as Beta-methyl amino acid, C Alpha-Methyl amino acid and N Alpha-Methyl amino acid etc.) of D-amino acid (it has resistance to L-amino acid specific protease), D-and the amino acid whose combination of L-, beta amino acids or various other design or non-natural existence, thereby gives specific character.Synthetic amino acid can comprise the ornithine of corresponding Methionin and the nor-leucine of corresponding leucine or Isoleucine.In addition, described polypeptide can have the plan peptide bond, ester bond for example, thus preparation has the polypeptide of new property.For example, can generate and introduce reduction peptide bond (that is, R ₁-CH ₂-NH-R ₂, R wherein ₁and R ₂for amino-acid residue or sequence) polypeptide.The reduction peptide bond can be introduced as the dipeptides subunit.This peptide species has resistance to protease activity, and has in vivo the transformation period of prolongation.Polypeptide can also comprise class peptide (glycine that N-replaces), and wherein side chain is connected on the nitrogen-atoms on molecular backbone chain, and not as be connected with alpha-carbon in amino acid.In the application's full text, polypeptide and peptide intention is used interchangeably, that is, in the situation that use the term peptide, it can also comprise polypeptide, and in the situation that use the term polypeptide, it also can comprise peptide.

Can use wedding agent to separate, catch or detect vesica.Described wedding agent can be the material in conjunction with vesica " house keeping protein (housekeeping protein) " or general vesica biomarker.Described biomarker can be CD63, CD9, CD81, CD82, CD37, CD53, Rab-5b, annexin V or MFG-E8.Four transmembrane proteins, have the membranin family of four membrane spaning domains, can be used as general vesica mark.Described four transmembrane proteins comprise CD151, CD53, CD37, CD82, CD81, CD9 and CD63.Identified in Mammals and surpassed 30 kind of four transmembrane protein, it comprises TSPAN1 (TSP-1), TSPAN2 (TSP-2), TSPAN3 (TSP-3), TSPAN4 (TSP-4, NAG-2), TSPAN5 (TSP-5), TSPAN6 (TSP-6), TSPAN7 (CD231, TALLA-1, A15), TSPAN8 (CO-029), TSPAN9 (NET-5), TSPAN10 (depending on fibroin (Oculospanin)), TSPAN11 (CD151 sample), TSPAN12 (NET-2), TSPAN13 (NET-6), TSPAN14, TSPAN15 (NET-7), TSPAN16 (TM4-B), TSPAN17, TSPAN18, TSPAN19, TSPAN20 (UP1b, UPK1B), TSPAN21 (UP1a, UPK1A), TSPAN22 (RDS, PRPH2), TSPAN23 (ROM1), TSPAN24 (CD151), TSPAN25 (CD53), TSPAN26 (CD37), TSPAN27 (CD82), TSPAN28 (CD81), TSPAN29 (CD9), TSPAN30 (CD63), TSPAN31 (SAS), TSPAN32 (TSSC6), TSPAN33 and TSPAN34.Other common vesica mark comprises the mark of listing in table 3.Can use any these protein as the vesica mark.

Table 3: the protein of observing in the vesica from the many cells type

Described wedding agent can also be in conjunction be derived from particular cell types (such as tumour cell) (as, for the wedding agent of tissue factor, EpCam, B7H3 or CD24) or the material of the vesica in specific cells source.For separating of or the wedding agent that detects vesica can be for the wedding agent that is selected from the antigen in Fig. 1.The wedding agent of vesica also can be selected from the wedding agent of listing in Fig. 2.Described wedding agent can be for antigen, such as four transmembrane proteins, MFG-E8, annexin V, 5T4, B7H3, caveolin (caveolin), CD63, CD9, CAM 120/80, tissue factor, MFG-E8, TMEM211, CD24, PSCA, PCSA, PSMA, Rab-5B, STEAP, TNFR1, CD81, EpCam, CD59, CD81, ICAM, EGFR or CD66.For hematoblastic wedding agent, can be glycoprotein, such as GpIa-IIa, GpIIb-IIIa, GpIIIb, GpIb or GpIX.Can use one or more wedding agents (for example for one or more wedding agents of two or more antigens) to separate or detect vesica.Can be according to separating or detecting and select wedding agent used derived from the vesica of particular cell types or the needs of cell source specificity vesica.

Can use one or more wedding agents (for example for one or more wedding agents of two or more antigens) to separate or detect vesica.Can select wedding agent used from the vesica of particular cell types or the needs of cell source specificity vesica according to separation or detection resources.

Wedding agent can also directly or indirectly be connected with solid surface or substrate.But solid surface or substrate can be wedding agent can be directly or indirectly attached the solid of any physical sepn, include but not limited to the surface that for example, pearl by microarray and hole, particle (pearl), post, optical fiber, swab, glass and glass modification or functionalization, quartz, mica, diazotizing film (paper or nylon), polyoxymethylene, Mierocrystalline cellulose, cellulose acetate, paper, pottery, metal, metalloid, semiconductor material, quantum dot, coating or particle, other chromatographic material, magnetic-particle provide; Plastics (multipolymer, polypropylene, polyethylene, polybutene, Polyurethanes, the TEFLON that comprise acrylic acid or the like, polystyrene, vinylbenzene or other material ^tMdeng), the surface that provides of polysaccharide, nylon or Nitrocellulose, resin, silicon-dioxide or the material based on silicon-dioxide (comprising silicon and modified silicon), carbon, metal, unorganic glass, plastics, pottery, conductive polymers (comprising such as polypyrrole and the such polymkeric substance of poly-indoles); The surface of microstructure or nanostructure, the surface that for example array of nucleic acid bedding, nanotube, nano wire or nano particle are decorated; Perhaps porous surface or gel, for example methacrylic ester, acrylamide, glycopolymers, Mierocrystalline cellulose, silicate or other fibrous or chain polymer.In addition, as known in the art, can use the passivation of (comprising polymkeric substance, as dextran, acrylamide, gelatin or agarose) that there is various material or the coating of chemical derivatization to carry out coated substrates.These coatings can contribute to array for biological sample.

For example, can be incorporated into solid substrate as hole for separating of the antibody of vesica, for example, as commercially available flat board (deriving from Nunc, Milan, Italy).Can use antibody to apply each hole.In some embodiments, for separating of the antibody of vesica, with the solid substrate such as array, be combined.Described array can have predetermined interaction of molecules, in conjunction with the spatial disposition in island, biomolecules, Dai, territory, district, or in conjunction with island or be distributed in the spatial disposition of the wedding agent in zone of dispersion.In addition, the term array can be used in reference to from the teeth outwards many arrays of arranging in this article, can be for example the situation of a plurality of copies of having array on surface.This surface with many arrays can also be called poly array or repeat array.

Wedding agent can also be combined with particle, for example pearl or microballoon.For example, the vesica composition is had to specific antibody can be combined with particle, and the particle of antibodies can be for separating vesica from biological sample.In some embodiments, described microballoon can be for magnetic or fluorescently-labeled.In addition, the wedding agent for separating of vesica can be solid substrate itself.For example, can use latex beads, for example aldehyde/vitriol pearl (Interfacial Dynamics, Portland, OR).

In addition, can also separate vesica with the wedding agent of being combined with magnetic bead.For example, can collect biological sample (for example serum) from the patient with for colon cancer screening.Described sample can be hatched with together with anti-CCSA-3 (colorectal carcinoma specific antigens) on being coupled to the magnetic microballon.Low-density microtrabeculae can be placed in the magnetic field of MACS separator, then uses buffered soln (for example Tris buffer saline) to wash this post.Then, the magnetic immuno mixture is applied on post, and abandons unconjugated nonspecific material.Can reclaim the vesica that CCSA-3 selects by removing post and place it in collection tube from separator.Buffer reagent can be joined on described post, and can be by applying the vesica that magnetic mark is provided with the plunger provided together with this post.Can in the IgG elution buffer, dilute the vesica separated, thereby then microballon be separated with vesica by mixture is centrifugal.Can be by the pellet resuspended of the cell source specificity vesica that separates for example, in damping fluid (phosphate buffered saline (PBS)), and carry out quantitatively.Perhaps, due to the cell source specificity vesica of antibody capture and the strong adsorptive power between the magnetic microballon, the vesica that can use proteolytic ferment (for example trypsinase) to discharge to catch and without centrifugal.Can together with the cell source specificity vesica of proteolytic ferment and antibody capture, hatch the time that is enough to discharge described vesica.

For example, for separating of the wedding agent (antibody) of vesica, preferably with the biological sample contact that comprises the target vesica, continue at least to be enough to make described wedding agent to be combined time of composition of described vesica.For example, antibody can contact with biological sample the various time periods by several seconds to a couple of days, includes but not limited to approximately 10 minutes, 30 minutes, 1 hour, 3 hours, 5 hours, 7 hours, 10 hours, 15 hours, 1 day, 3 days, 7 days or 10 days.

Wedding agent (for example antigen listed in Fig. 1 being had to wedding agent listed in specific antibody or Fig. 2) can be with including but not limited to following material mark: magnetic mark thing, fluorescence part, enzyme, chemiluminescence probe, metallic particles, nonmetal colloidal particle, polymeric dye particles, pigment molecule, granules of pigments, electroactive substance, semiconductor nanocrystal or other nano particle (comprising quantum dot or gold grain).Described marker can for but be not limited to fluorophore, quantum dot or radioactively labelled substance.For example, described marker can be radio isotope (radionuclide), for example ³h, ¹¹c, ¹⁴c, ¹⁸f, ³²p, ³⁵s, ⁶⁴cu, ⁶⁸ga, ⁸⁶y, ⁹⁹tc, ¹¹¹in, ¹²³i, ¹²⁴i, ¹²⁵i, ¹³¹i, ¹³³xe, ¹⁷⁷lu, ²¹¹at or ²¹³bi.Described marker can be fluorescent marker, for example Rare Earth Chelate (europium inner complex); Fluoresceins, such as but not limited to FITC, CF, 6-Fluoresceincarboxylic acid; Rhodamine, such as but not limited to TAMRA; Dansyl; Liz amine (Lissamine); Anthocyanidin; Phycoerythrin; Texas Red; And their analogue.Described fluorescent marker can be one or more in FAM, dRHO, 5-FAM, 6FAM, dR6G, JOE, HEX, VIC, TET, dTAMRA, TAMRA, NED, dROX, PET, BHQ, Gold540 and LIZ.

Wedding agent is mark directly or indirectly, and for example, described marker is connected with antibody by vitamin H-streptavidin.Perhaps, the antibody un-marked, but subsequently first antibody and order how antigen be combined and contact with the second antibody of mark afterwards.

For example, various enzyme-substrate markers are available or are disclosed (for example, referring to U.S. Patent No. 4,275,149).The chemically changed of the common catalysis chromophoric substrate of enzyme, described change can be used various commercial measurements.For example, the colour-change that enzyme can catalytic substrate, it can carry out metric measurement.Perhaps, enzyme can change fluorescence or the chemoluminescence of substrate.The example of enzyme labelling thing comprises luciferase (for example Photinus pyralis LUC and bacterial luciferase; U.S. Patent No. 4,737,456), fluorescein, 2,3-dihydro phthalazine diketone, malate dehydrogenase (malic acid dehydrogenase), urase, peroxidase (such as horseradish peroxidase (HRP)), alkaline phosphatase (AP), beta-galactosidase enzymes, glucoamylase, N,O-Diacetylmuramidase, carbohydrate oxidase (such as glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase (G6PD)), heterocycle oxydase (such as uriKoxidase and XOD), lactoperoxidase, microperoxisome etc.The example of enzyme-substrate combination includes but not limited to: use the horseradish peroxidase (HRP) of catalase as substrate, catalase oxidation dye precursors (O-Phenylene Diamine (OPD) or 3 for example wherein, 3 ', 5,5 '-tetramethyl biphenyl amine hydrochlorate (TMB)); Use the alkaline phosphatase (AP) of p-nitrophenyl phosphate as the color bodies substrate; And the beta-D-galactosidase (β-D-Gal) that uses color bodies substrate (for example p-nitrophenyl-beta-D-galactosidase) or the substrate that fluoresces (4-methyl umbrella shape base-beta-D-galactosidase).

According to separation used or detection method, described wedding agent can for example, be connected with solid surface or substrate (array, particle, hole and other substrate mentioned above).For the method for antibody and the direct chemical coupling of cell surface is known in the art, and can comprise that the antibody that (for example) used glutaraldehyde or maleimide to activate carries out coupling.Comprise that for the method for using multistep process to carry out chemical coupling the succinimide ester of biotinylation, use (for example) trinitrophenol (TNP) or digoxigenin carrys out these compounds of coupling.Can for example, by (), with Bio base-N-hydroxy-succinamide, complete biotinylation.The succinimide group, and preferably reacts with amino group to the condition between about pH 8.5 at about pH 8.0 higher than 7 effectively in the pH value.Then can for example, by (), use dithiothreitol (DTT) to process cell adds the vitamin H maleimide to complete biotinylation.

Flow cytometry

Can also implement to use particle as separation or the detection to vesica of pearl or microballoon with flow cytometry.Can come sorting to be suspended in the microscopic particles in fluid stream with flow cytometry.When particle by the time, they are can be optionally charged and when they leave, can deflect into independently in flow path.Therefore, likely with the accuracy of height, separate the colony from original stock (biological example sample) with speed.Flow cytometry allows single celled physics and/or the chemical feature that flows through optics/electronic detecting device carried out to multiparametric analysis simultaneously.The light beam (being generally laser) of monofrequency (color) points on the fluid stream focused at hydrokinetics.A plurality of detectors aim at the point of fluid stream through light beam; One overlaps (direct scattering or FSC) and several perpendicular to this light beam (lateral scattering or SSC) and one or more fluorimetric detectors with this light beam.

Each suspended particle by light beam is scattered beam in some way, and the light lower than light source with transmitting frequency that can be excited of the fluorescence chemical material in particle.This combination of scattered light and fluorescence is picked up by detector, and locates the fluctuation of brightness by analyzing each detector (detector of each fluorescence emission peak), likely infers the various factors about the physics and chemistry structure of each individual particle.FSC is relevant to cell size, and SSC depends on the inside complicacy of particulate, for example the roughness of the amount of nuclear shape, cytoplasmic granule thing and type or film.Some flow cytometers are only measured with scattering of light without fluorescence.

Flow cytometer can be analyzed several thousand particles with the mode per second of " in real time ", and can separate on one's own initiative and separate the particle with specified properties.They provide in each analysis phase for the high-throughout automatization of the setup parameter of a large amount of single cells quantitatively with separate.Flow cytometer can have a plurality of laser and fluorimetric detector, thereby allows to use multiple marker more accurately it to be specified with the phenotype according to target group.Therefore, flow cytometer (such as the polychrome flow cytometer) can be used for detecting one or more vesicas with multiple fluorescent mark or color.In some embodiments, but the also sorting or separate different vesica colony of described flow cytometer, such as according to size or according to different marks.

Described flow cytometer can have one or more laser, such as 1,2,3,4,5,6,7,8,9,10 or more kinds of laser.In some embodiments, described flow cytometer can detect more than a kind of color or fluorescent mark, such as at least 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19 or 20 kind of different colours or fluorescent mark.For example, described flow cytometer can have at least 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19 or 20 fluorimetric detectors.

Can be used for detecting or analyze one or more vesicas, for sorting or the example that separates the flow cytometer be obtained commercially of different vesica colonies, include but not limited to MoFlo ^tMxDP Cell Sorter (Beckman Coulter, Brea, CA), MoFlo ^tMlegacy Cell Sorter (Beckman Coulter, Brea, CA), BD FACSAria ^tMcell Sorter (BD Biosciences, San Jose, CA), BD ^tMlSRII (BD Biosciences, San Jose, CA) and BD FACSCalibur ^tM(BD Biosciences, San Jose, CA).According to what hereinafter further describe, use polychrome or many fluorocytes calculating instrument to can be used for the multiple analysis of vesica.In some embodiments, but described flow cytometer sorting, therefore and surpass a kind of vesica colony according to one or more feature collection or sorting.For example, two kinds of vesica colonies are different in size, thereby make the vesica in each colony have similar magnitude range and can be detected or sorting by difference ground.In another embodiment, two kinds of different vesica colonies carry out the difference mark.

Thereby the data that derive from flow cytometer can be mapped and obtained histogram in the mode of 1 dimension, or in the mode of 2 dimensions as scattergram or use newer software to map in 3 dimension modes.Zone on these aspects can come order to separate by a series of inferior extraction steps (it is called gate).There is the specific gate scheme for diagnosis and clinical purpose, particularly relate to hematology.These mappings complete with logarithmic scale usually.Because the emission spectrum of different fluorescence dyes is overlapping, so have to compensate by electricity and account form at the signal at detector place.Fluorophore for the mark biomarker can comprise Ormerod, Flow Cytometry the 2nd edition, Springer-Verlag, New York (1999) and Nida etc., Gynecologic Oncology 2005; Fluorophore described in 4 889-894, it is incorporated to this paper by reference.

Many complex analyses

Multiple experiment comprises can measure the experiment of multiple analytes in single analysis simultaneously.Can assess vesica and associated biomolecule mark in multiple mode.Different wedding agents can be for multiplexing different vesica colony.Can separate or detect different vesica colonies with different wedding agents.Can use different signal tracers (for example fluorophore, quantum dot or radioactively labelled substance, each colony in the mark biological sample as described above).Described marker can with the direct coupling of wedding agent, or indirectly for detection of the wedding agent in conjunction with vesica.The Population detected in multiple analysis depends on the resolving power of marker and the summation of signal, because can produce the signal of summation with the vesica colony of the two or more difference mark of two or more affine combination of elements.

Can carry out at least 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,25,50,75 or 100 kind of different vesica colony multiplexing.For example, cell source is had to specific a kind of vesica colony can be analyzed together from different cell sources being had to specific the second vesica colony, and wherein each colony is with different marker marks.Perhaps, the vesica colony that has specific biomarker or the biological marking can analyze together from the second vesica colony with different biomarkers or biological marking.In some cases, can in single analysis, assess hundreds of or thousands of kinds of vesicas.

In one embodiment, for example be applied to, in a plurality of substrates (pearl) and carry out multiple analysis by comprising multiple vesica more than a kind of vesica colony.Each pearl and one or more trapping agent couplings.A plurality of pearls are divided into to subgroup, and the pearl that wherein has identical trapping agent or trapping agent combination forms the subgroup of pearl, thereby makes each subgroup of pearl have trapping agent or the trapping agent combination that is different from another pearl subgroup.Then, described pearl can be for catching the vesica that comprises the composition combined with described trapping agent.Described different subgroup can be for catching different vesica colonies.Then, the vesica that can catch by detecting one or more biomarker analyses.

Flow cytometer can with based on particle or the analytical procedure based on pearl, be used in combination.Can use multiparameter immune analysis method or other high-throughout determination method (reporter molecules that its use has the pearl coating of homology aglucon and has the specific activity compatible with the height sensitivity automatic technology).For example, the pearl of the pearl in each subgroup and another subgroup is the difference mark.In the analytical system based on particle, for example, for wedding agent or the trapping agent (capture antibody) of vesica, can be fixed in addressable pearl or microballoon.For each wedding agent of each single binding analysis (for example immunoassay when wedding agent is antibody) can with the microballoon of completely different type (, microballon) coupling, and binding analysis reacts and occurs on the surface of microballoon.Microballoon can distinguish according to different markers, for example from the another kind of microballoon with different trapping agents, compares, and the microballoon with particular capture agent has different signal tracers.For example, microballoon can dye as having discrete fluorescence intensity, thereby makes the fluorescence intensity of the microballoon with particular combination agent be different from the fluorescence intensity of the another kind of microballoon with different wedding agents.

Described microballoon can use at least 2 kinds of different markers or dyestuff to carry out mark or dyeing.In some embodiments, use at least 3,4,5,6,7,8,9 or 10 kind of different marker carry out mark to described microballoon.Different microballoons in multiple microballoon can have more than a kind of marker or dyestuff, and wherein various microballoon subgroups have marker or the dyestuff of different ratios and composition, thereby can detect the different microballoons with different wedding agents.For example, the marker of various different ratioss and composition and dyestuff can allow different fluorescence intensities.Perhaps, various different ratioss and composition can be for generation of different detecting patterns to identify wedding agent.Described microballoon can carry out external marking or dyeing, or can have inherent fluorescence or signal mark.Pearl can load their suitable wedding agents individually, and therefore can separate different vesica colonies according to the different wedding agents on the microballoon (different wedding agents and its coupling) of difference mark.

In another embodiment, can use planar substrates to carry out multiple analysis, wherein said substrate comprises multiple trapping agent.Described multiple trapping agent can be caught one or more vesica colonies, and detects one or more biomarkers of the vesica of catching.According to what hereinafter further describe, planar substrates can be microarray or other substrate.

New junction agent

Can use wedding agent for the novel components of the vesica antibody of the specific neoantigen of target vesica (for example for) to separate or detect vesica.For example can use, from the different test compounds of the known composition of substrate (array or a plurality of particle) combination and separate or differentiate the specific neoantigen of target vesica, it can allow only to use the small portion space and a large amount of chemistry/structure spaces is fully sampled.The biomarker that the neoantigen of identifying also can be used as described vesica plays a role.For example, the neoantigen of identifying for the specific vesica of cell source can be useful biomarker.

Can identify wedding agent by or vesica of heterogeneous colony homogeneous for test compounds screening.Because the composition of each test compounds on substrate surface is known, so this has formed the screening to the affinity element.For example, the specific location of test compounds array on the substrate addressable locations comprises test compounds, and can be for the identification of one or more wedding agents for vesica.Less variation based on core sequence or structure, test compounds can be all incoherent or relevant.Different test compounds for example can comprise, on the variant (shaped body of polypeptide), structure of given test compounds or form upper incoherent test compounds or their combination.

Test compounds can be class peptide, polysaccharide, organic compound, mineral compound, polymkeric substance, lipid, nucleic acid, polypeptide, antibody, protein, polysaccharide or other compound.Described test compounds can be for natural or synthetic.Described test compounds can comprise based on multiple key or key combination (such as acid amides, ester, ether, mercaptan, free radical addition, metal-complexing etc.) thus linearity or heteropolymerization compound, branched structure, ring texture, the cavity configuration of branch or there is other structure that a plurality of connection site of closing on play the support effect when carrying out specific addition, or formed by them.Can use standard method in this area by the test compounds point sample in substrate or to carry out original position synthetic.In addition, it is synthetic to detect useful interaction that described test compounds can be carried out point sample or original position with array mode, for example collaborative combination.

Described test compounds can, for having the polypeptide of known aminoacid sequence, therefore, detect the evaluation of the polypeptide that can realize the known amino acid sequence to being used as wedding agent to the combination of test compounds and vesica.For example, the homogeneous colony of vesica can be applied on the sample application array on slide glass (comprising several to 1,000,000 kind of test polypeptide with variable amino acid length).Described polypeptide can be connected with surface by the C-end.Described peptide sequence can generate by 19 seed amino acids (not comprising halfcystine) are random.Association reaction can comprise nonspecific competitor (for example using the excessive bacterioprotein of another kind of dye marker), thereby can measure the specificity ratio in conjunction with target for each polypeptide.Can select to have the polypeptide of high specific and combination.On each point, the identity of polypeptide is known, and can easily identify thus.Once identify, described homogeneous vesica colony (for example cell source specificity vesica) is had to specific neoantigen, in described method, can use these antigens to separate this class cell source specificity vesica hereinafter subsequently.

In addition, can also identify the antibody as the vesica wedding agent with array.Test antibody can be connected with array, and is screened for heterogeneous vesica colony, with identify can for separating of or identify the antibody of vesica.In addition, can also screen with antibody array the vesica colony (for example cell source specificity vesica) of homogeneous.To separate or to detect the vesica colony of homogeneous, can also identify that the colony to homogeneous has specific one or more protein biomarkers except identifying antibody.Can use commercially available platform, it has the test antibody of the preliminary election that is connected in described array or the test antibody that customization is selected.For example, can screen with the vesica in prostate cancer cell source the antibody array from Full Moon Biosystems, it will be wedding agent (for example, referring to Figure 62) for the Identification of the antibodies of Bcl-XL, ERCC1, keratin 15, CD81/TAPA-1, CD9, epithelial specific antigen (ESA) and mastocyte Chymotrypsin, and the protein of identifying can be as the biomarker of described vesica.

Can also identify stand-by antibody or the synthetic antibody of making wedding agent by the peptide array.Another kind method is to use by antibody phage to show and produce synthetic antibody.The M13 phage library of antibody (for example Fab) is presented on the surface of phage particle as the syzygy of coat protein.Each phage particle presents unique antibody, and has sealed the carrier that comprises coding DNA.Can build highly various library, and mean as phage library, it can be for selecting the antibody in conjunction with fixing antigen.Fixing antigen has kept antigen in conjunction with phage, and unconjugated phage is removed by washing.The phage library that can increase and keep by the infection of escherichia coli host, and the storehouse of amplification can be used for to the selection of other some rounds, thus finally obtain antigen in conjunction with the dominant colony of clone.At this one-phase, can separate single phage clone and carry out DNA sequencing, thus the sequence that the antibody presented is taken out in decoding.By using phage display and other method as known in the art, can produce the high-affinity designerantibodies for vesica.

In addition, the analysis based on pearl can also be for the identification of new junction agent to separate or the detection vesica.Test antibody or peptide can with the particle coupling.For example, pearl can with antibody or peptide coupling, and for detection of the protein with quantitatively expressing on the surface of vesica colony, thus find and select specifically can target from the novel antibody of the vesica of particular organization or tumor type.Any molecule that organ source (organic origin) can be provided by the specification sheets that uses commercially available test kit to provide according to manufacturers successfully with the polystyrene bead coupling.The group of each pearl can be with specific detectable wavelength color development, and each group can be connected with known antibody or peptide, wherein said antibody or peptide can be connected with exosome protein for measuring specifically which pearl, thereby are complementary with the antibody of coupling before or the epi-position of peptide.Described pearl can be discrete fluorescence intensity dyeing, make each pearl with varying strength there are different wedding agents mentioned above.

For example, rule of thumb definite performance analysis scope can be diluted to suitable concentration by the vesicle formation of purifying in analysis buffer.The coupling pearl that can prepare sufficient volume, and the antibody coupling pearl decile of about 1 μ l to hole neutralization is adjusted to approximately 50 μ l of final volume.Once the pearl of antibody coupling be joined in the vacuum compatible plate, can be washed to guarantee suitable conjugation condition to this pearl.Then, the vesicle formation of appropriate volume can be joined in each hole to be determined and hatch described mixture, for example 15-18 hour.Can use detection antibody dilution solution to prepare the detection antibody of sufficient volume, and hatch 1 hour with described mixture or the required time.Subsequently, washed described pearl before adding detection antibody (expression vitamin H) mixture formed by the streptavidin phycoerythrin.Then, can wash pearl, and vacuum take-off several times, then use the software provided together with instrument to be analyzed on the suspension array system.Afterwards, can illustrate from this analysis the identity of the antigen that can be used for optionally extracting vesica.

Used imaging system analysis can for detection of and the protein of quantitatively expressing on the surface of vesica, thereby find and select specifically and enrichment from the vesica of particular organization, cell or tumor type.Can use antibody, peptide or cell with the coupling of porous composite carbon coated board.The capture antibody that can list on the carbon working-surface of patterning by use is measured the multiple analytes in hole simultaneously.Then, can be in the electrode hole with enhancing electricity-Chemiluminescent plate, applying marking has the antibody test analyte of reagent.Any molecule in organ source can be successfully and the coupling of described carbon coated board.Can go out the protein of expressing on the vesica surface by this Analysis and Identification, and can be using it as target for select specifically and enrichment from the vesica of particular organization or tumor type.

Described wedding agent can also be for fit, and it refers to the nucleic acid of can the molecule outside its complementary sequence being combined.Fitly generally comprise 30-80 nucleic acid and there is the high-affinity (K reported for specific target molecule _dbetween 10 ^-11-10 ^-6mole/l).Can use fit (the Tuerk &amp of Fas lignand system evolution (SELEX) technical evaluation of index concentration for target; Gold, Science 249:505-510,1990; Ellington & Szostak, Nature 346:818-822,1990), for example, in U.S. Patent No. 5,270,163,6,482,594,6,291,184,6,376,190 and US 6,458, described in 539.Nucleic acid library can be contacted with the target vesica, and those nucleic acid that will be combined with described target specifically remaining nucleic acid (its can not be combined specifically described target) in library separates.By the nucleic acid amplification that separates to produce part enrichment storehouse.Combination of many wheels, separately and amplification (that is, selection) realized thering are one or more fit evaluations of required activity.Thereby for the identification of the another kind of method of fit separation vesica, in U.S. Patent No. 6,376, describe to some extent in 190, described document description increasing or reduction in conjunction with the frequency that makes this nucleic acid of the peptide by library amplifying nucleic acid and chemosynthesis.Can also use improved method, for example Laser SELEX or the deSELEX described in the open No.20090264508 of United States Patent (USP).

Microfluidic device

For separating of or identify that the method for vesica can be used in combination with microfluidic device.Can use microfluidic device to implement all methods of separating or detect as described herein vesica.The microfluidic device that also can be described as " chip lab " system, biomedical microelectromechanical systems (bioMEM) or multi-part integrated system can be used for separating and analyzing vesica.This type systematic makes process and other process microminiaturization and the compartmentation that allows vesica combination, biomarker to detect.

Microfluidic device can also be for selecting to separate vesica by difference in size or affinity.For example, microfluidic device can be used for using for separate one or more passages of vesica from biological sample from one or more wedding agents of biological sample separation vesica according to size or by use.Biological sample can be incorporated in one or more microfluidic channel, it optionally allows vesica to pass through.Can for example, according to the character (size of vesica, shape, deformability or the biological marking) of vesica, be selected.

In one embodiment, the heterogeneous population of vesica can be incorporated in microfluidic device, and can obtain one or more different homogeneous vesica colonies.For example, different passages can have that different size is selected or wedding agent to select different vesica colonies.Therefore, microfluidic device can separate multiple vesica, the biological marking that wherein at least one subgroup of this multiple vesica comprises another subgroup that is different from described multiple vesica.For example, described microfluidic device can separate at least 2,3,4,5,6,7,8,9,10,15,20,25,30,40,50,60,70,80,90 or 100 different vesica subgroups, and wherein each vesica subgroup comprises the different biological markings.

In some embodiments, described microfluidic device can comprise the further enrichment that allows vesica or one or more passages of selection.The vesica colony of enrichment after by first channel be directed in second passage, and it allows required vesica or vesica colony to pass through with further enrichment, for example, through being present in one or more wedding agents in second passage.

Can together with microfluidic device, use analysis and the analysis based on pearl based on array.For example, wedding agent can with the pearl coupling, and can in microfluidic device, implement the association reaction between described pearl and vesica.In addition, can also use microfluidic device to carry out multiplexing.Different compartments can comprise the different wedding agents for different vesica colonies, and wherein each colony is the specific vesica of different cell sources colony.In one embodiment, each colony has the different biological markings.Can in microfluidic device, implement the hybridization between microballoon and vesica, and reaction mixture can be delivered in proofing unit by defeated.Described proofing unit (for example two or many laser detection systems) can be the part of described microfluid system, and can identify each pearl or microballoon with laser by the color-code of pearl or microballoon, and another Shu Jiguang can detect the hybridization signal relevant to each pearl.

Can be used for vesica or the example through adapting to the microfluidic device for vesica includes but not limited to be described in U.S. Patent No. 7,591,936, 7,581,429, 7,579,136, 7,575,722, 7,568,399, 7,552,741, 7,544,506, 7,541,578, 7,518,726, 7,488,596, 7,485,214, 7,467,928, 7,452,713, 7,452,509, 7,449,096, 7,431,887, 7,422,725, 7,422,669, 7,419,822, 7,419,639, 7,413,709, 7,411,184, 7,402,229, 7,390,463, 7,381,471, 7,357,864, 7,351,592, 7,351,380, 7,338,637, 7,329,391, 7,323,140, 7,261,824, 7,258,837, 7,253,003, 7,238,324, 7,238,255, 7,233,865, 7,229,538, 7,201,881, 7,195,986, 7,189,581, 7,189,580, 7,189,368, 7,141,978, 7,138,062, 7,135,147, 7,125,711, 7,118,910, 7,118,661, 7,640,947, 7,666,361, 7,704,735, and those devices in International Patent Publication No. WO 2010/072410, each patent or application are incorporated to this paper in full by reference.Another example for the disclosed method of the present invention is described in Chen etc., " Microfluidic isolation and transcriptome analysis of serum vesicles; " Lab on a Chip, on December 8th, 2009 DOI:10.1039/b916199f.

In one embodiment, for separating of or the microfluidic device that detects vesica comprise on width in approximately 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,35,40,45,50,55 or 60mm or the passage of width between 2-60,3-50,3-40,3-30,3-20 or 4-20mm.Microchannel can have and is less than about 10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,45,50,55,60,65 or 70 μ m or between the degree of depth of about 10-70,10-40,15-35 or 20-30 μ m.In addition, microchannel has and is less than approximately 1,2,3,3.5,4,4.5,5,5.5,6,6.5,7,7.5,8,8.5,9,9.5 or the length of 10cm.Described microfluidic device can have groove on its top board, and its width is less than approximately 40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,65,70,75 or 80 μ m or between about 40-80,40-70,40-60 or 45-55 μ m.The degree of depth of described groove can be less than approximately 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,25,30,35,40,45 or 50 μ m, as between about 1-50,5-40,5-30,3-20 or 5-15 μ m.

Described microfluidic device can have and is connected in channel surface or is present in one or more wedding agents in passage.For example, described microchannel can have one or more trapping agents, such as the trapping agent for EpCam, CD9, PCSA, CD63, CD81, PSMA, B7H3, PSCA, ICAM, STEAP and EGFR.In one embodiment, microchannel surface is processed with avidin, and can be to injecting in described passage through biotinylated trapping agent (such as antibody) with in conjunction with avidin.

Biological sample can be flowed in described microfluidic device or microchannel, its flow velocity is as at least about 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,25,30,35,40,45 or 50 μ l per minutes, as between about 1-50,5-40,5-30,3-20 or 5-15 μ l per minute.In described microfluidic device, can catch and one or more vesicas of direct-detection.The vesica of perhaps, catching can discharge and leave described microfluidic device before analysis.In another embodiment, one or more captive vesicas of cracking analytical pyrolysis thing in described microchannel.Can make lysis buffer flow through the vesica that described passage cracking are caught.For example, lysis buffer can be flowed into to described device or microchannel, its flow velocity is as at least about 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,25,26,27,28,29,30,35,40,45 or 50 μ l per minutes, as between about 1-50,5-40,10-30,5-30 or 10-35 μ l per minute.Can collect and analyze described lysate, such as carrying out RT-PCR, PCR, mass spectrum, Western trace or other analyzes to detect one or more biomarkers of described vesica.

Cell source and disease specific vesica

Wedding agent disclosed herein can for separating of or detect vesica, cell source vesica or there is the vesica of the particular organisms marking for example.Described wedding agent can be used for from sample separation or detects heterogeneous vesica colony or can be used for from heterogeneous vesica colony's separation or detect homogeneous vesica colony, for example has the cell source specificity vesica of the particular organisms marking.

Can analyze homogeneous vesica colony (such as cell source specificity vesica) and for characterizing experimenter's phenotype.Cell source specificity vesica is the vesica that is derived from particular cell types, and it can be including but not limited to the cell of particular organization, from cell, circulating tumor cell or the parent of specific target tumor or target disease tissue or the cell of fetal origin.Described vesica can be derived from tumour cell or pneumonocyte, pancreatic cell, gastric cells, intestinal cells, bladder cell, nephrocyte, gonad cell, testicular cell, skin cells, colorectal cell, mammary gland cell, prostatic cell, brain cell, oesophagus cell, liver cell, placenta cells or fetal cell.The vesica of described separation can also be from specific sample type, and for example allantois steeps.

The cell source specificity vesica of biological sample can separate with cell source being had to specific one or more wedding agents.The vesica that is used for analysis of disease or situation can have with the biomarker to this disease or situation specific one or more wedding agents to be separated.

Before cell source specificity vesica is carried out to separation and detection, can be according to mentioned above that vesica is concentrated, such as by centrifugal, chromatogram or filtration, thereby obtained heterogeneous vesica colony before the specificity vesica of isolated cell source.Perhaps, before the vesica of isolated cell source, described vesica is without concentrated, or described biological sample does not carry out enrichment for vesica.

Figure 61 B illustrated describe for separating of or the schema of the method 6100B of identification of cell source specificity vesica.At first, in step 6102, by the experimenter, obtain biological sample.Described sample can derive from the third party, or derives from the same side who implements described analysis.Then, in step 6104 from biological sample isolated cell source specificity vesica.Analyze subsequently separated cell source specificity vesica in step 6106, and come identification of organism mark or the biological marking for specific phenotype in step 6108.Described method can be for multiple phenotype.In some embodiments, before step 6104, that vesica is concentrated or separate by biological sample, thus obtain the vesica colony of homogeneous.For example, can separate heterogeneous vesica colony by centrifugal, chromatogram, filtration or other method mentioned above, then use especially for separating of or identify one or more wedding agents of the vesica that is derived from particular cell types.

Can carry out the biological sample isolated cell source specificity vesica from the experimenter by using one or more wedding agents that combine with cell source specificity vesica with specificity highly.In some cases, can carry out isolated cell source specificity vesica with single wedding agent.In other situation, can carry out with the combination of wedding agent isolated cell source specificity vesica.For example, can with at least 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,25,50,75 or 100 kind of different wedding agent carry out the vesica in isolated cell source.Therefore, can for example, by identify vesica colony (vesica that there is identical wedding agent spectrum) with single or multiple wedding agent.

Can select these wedding agents for for example, specificity for the specific target antigen of cell source (people's tumour, autoimmune disease, cardiovascular disorder, sacred disease, infection or Other diseases or the relevant cell source of lacking of proper care) according to one or more wedding agents.Described cell source can be from for diagnosis, prognosis, disease classification, treatment diagnosis, respondent/non-respondent's state anticipation, diseases monitoring, treatment monitoring etc., providing these diseases or the cell of the relevant information of lacking of proper care.Described cell source can also carry out to have by oneself the cell that can be used for finding for biomarker wherein.Can use separately or use in combination with the unrestricted example of the antigen of isolated cell source specificity vesica, disease specific vesica or tumour-specific vesica as shown in Figure 1, and also will be described hereinafter.Described antigen can comprise the film conjugated antigen that wedding agent can contact.Described antigen can be the biomarker with characterizing phenotypic correlation.

Those skilled in the art should understand, and the present invention includes any applicable antibody that can be used for the separate information vesica.Summarize according to this paper, can select the wedding agent of identified surface antigen and/or its fragment, as antibody, fit and lectin.Described wedding agent can be identified the antigen for required cell type or location specificity, and/or the identification biomarker relevant to required cell.Described cell can be, for example, tumour cell, other diseased cells, is used as the cell of disease marker, such as the immunocyte of activation etc.Those skilled in the art should understand, and for the wedding agent of arbitrary target cell, can be used for separating the vesica relevant to these cells.Those skilled in the art should be further appreciated that wedding agent disclosed herein can be used for detecting the target vesica.As unrestricted example, for the wedding agent of vesica biomarker directly or indirectly mark to detect the vesica of identical or different wedding agent institute combination by one or more.

Can be used in conjunction with the many targets to the wedding agent of cancer, autoimmune disease, cardiovascular disorder, sacred disease, infection or Other diseases or the relevant vesica of lacking of proper care shown in table 4.The vesica of the cell that one of the imbalance that can use one of antigen in this table to identify to be derived to listed is relevant.Described wedding agent (as antibody or fit) can be identified epi-position, its fragment of listed antigen, or wedding agent can be for suitable being used in combination arbitrarily.Also can identify other antigen relevant to described disease or imbalance to identify described vesica.Those skilled in the art should understand, the present invention includes any applicable antibody that can be used for the appreciation information vesica with for separating of, catch or detect, thereby identify vesica.

Table 4: for the identification of the illustrative antigen of various diseases and imbalance

According to method mentioned above, can carry out the specific vesica in isolated cell source with new junction agent.In addition, can also use the separation method of the Cell binding companion body based on these vesicas or wedding agent to come from the specific vesica in biological sample isolated cell source.These Cell binding companion bodys can include but not limited to peptide, protein, RNA, DNA, fit, cell or the relevant protein of serum, they only when having one or more specific biological marks in conjunction with these vesicas.Can be with single in conjunction with the companion body or wedding agent or implement separation or the detection of the specific vesica of cell source in conjunction with the combination of the companion body or wedding agent, being used singly or in combination of the described companion body or wedding agent can be carried out the specific separation of cell source or detection.The unrestricted example of these wedding agents provides in Fig. 2.For example, can use one or more wedding agents to separate for characterizing the vesica of mammary cancer, wherein said wedding agent includes but not limited to oestrogenic hormon, progesterone, He Saiting (Herceptin) (Herceptin), CCND1, MYC PNA, IGF-1PNA, MYC PNA, SC4 are fit (Ku), AII-7 is fit (ERB2), Gal-3, Saliva Orthana type O-glycan, L-PHA, half lactadherin-9 or their any combination.

Wedding agent can also be according to i) to the existence of the specific antigen of cell source specificity vesica tool, ii) to not the existing of the specific mark of the specific vesica tool of cell source, or iii) to the expression level of the specific biomarker of the specific vesica tool of cell source and for separating of or detect the specific vesica of cell source.Heterogeneous vesica colony can be put on the surface that is coated with specific-binding agent, wherein said wedding agent is designed to get rid of or confirm the cell source feature of vesica.Various wedding agents can be listed in solid surface or substrate as antibody, and can make described heterogeneous vesica colony and described solid surface or substrate contact be enough to occur the interactional time.Then, can be for the identification of the antigen-specific feature that given cell source is there is to specific vesica colony with the specific binding of described array surface or suprabasil given antibody location or non-specific binding.

According to mentioned above, can use magnetic catching method, fluorescence activated cell sorting (FACS) or laser cell counting to carry out the specific vesica of enrichment or isolated cell source with one or more wedding agents.The magnetic catching method can include but not limited to use magnetic activating cells sorter (MACS) microballon or magnetic post.Operable immunity is affine with the example of magnetic-particle method in U.S. Patent No. 4,551, describes to some extent in 435,4,795,698,4,925,788,5,108,933,5,186,827,5,200,084 or 5,158,871.Can also be according to U.S. Patent No. 7,399, the general method described in 632, carry out the specific vesica in isolated cell source by using the combination to the specific antigen of vesica tool.

With respect to biological sample, for separating of or additionally any other proper method of the described cell source specificity of enrichment vesica also can be used in combination with the present invention.For example, size exclusion chromatography (example gel infiltration post), centrifugal or density gradient centrifugation and filter method can be used with antigen selection Combination of Methods as herein described.Also can be according to Koga etc., Anticancer Research, 25:3703-3708 (2005), Taylor etc., Gynecologic Oncology, 110:13-21 (2008), Nanjee etc., Clin Chem, 2000; 46:207-223 or U.S. Patent No. 7,232, the method described in 653 is separated described cell source specificity vesica.

Therefore, the separable vesica gone out with the cellular segregation in the site of originated tumour or autoimmune disease, cardiovascular disorder, sacred disease, infection or Other diseases or imbalance.In some embodiments, the vesica separated is derived from and these diseases or the relevant cell of lacking of proper care, for example, the immunocyte played a role in the nosetiology of described disease, and provide diagnosis, prognosis, disease classification, treatment diagnosis, respondent/non-respondent's status predication, diseases monitoring, treatment monitoring etc. and these diseases or the relevant information of lacking of proper care to the analysis of described cell.Described vesica further can be used for finding biomarker.According to described herein, can assess subsequently the vesica of separation to characterize phenotype.

The assessment of vesica

Can by analyze from experimenter's biological sample and measure one or more vesica colonies in described sample level, amount or concentration and to characterizing described experimenter's phenotype.Can be before measuring the vesica amount purifying or concentrating vesicles.Perhaps, can be in the situation that without the vesica amount of purifying or concentrated direct analysis sample in advance.Described vesica can be cell source specificity vesica or has the vesica of the particular organisms marking.Can when characterizing phenotype (such as diagnosis, prognosis, treatment diagnosis or predicated response person/non-respondent's state), use this vesica amount.In some embodiments, described amount is for determining physiology or biological condition, such as gestation or pregnant stage.The vesica amount also can be used for determining the stage for the treatment of effect, disease or situation or the progress of disease or situation.For example, the vesica amount can be directly proportional or inverse ratio to going forward one by one of disease stage or process.The amount of vesica also can be used for progress or the reaction of monitoring experimenter to treating of monitoring of diseases or situation.

Can compare to assess vesica with reference level or the value of vesica by the level by vesica.Reference point can be specific for physics or end time.For example, reference point can be from the same experimenter with carrying out sample evaluating, or reference point can derive from representational sample colony (for example from the normal subjects who does not show disease symptoms sample).Therefore, reference point can provide the threshold measurement value that it can be compared with experimenter's sample reading of the vesica of analyzing in given sample colony.The data that can collect according to the sample of the many groups by corresponding to specific cohort are set this reference point, wherein said cohort include but not limited to age (for example adult of newborn infant, baby, teenager, youth, middle aged adult, old age and different ages), ethnic group/ethnic group, normal people to ill experimenter, smoker, non-smoker, the experimenter that receives treatment to untreated experimenter, different treatment times point or its combination with the individual or group of the particular subject of similar diagnosis or treatment.In addition, by particular individual being measured to the vesica level of difference treatment times point, can monitor described individuality to the reaction for the treatment of or the individual disease of being treated of monitoring or the progress of situation.

Reference point can be based on by identical the sample of experimenter's assessment, in order to a volume tracing is provided.Continually to the patient test can provide and reference point that particular patient set up before this between better comparison, and can make doctor disease stage or the process of assess patient more accurately, thereby provide information for better treatment decision-making.The individual intracellular vesicle level difference reduced can define specificity and personalized threshold value more for the patient.In temporal experimenter, difference makes each individual optimized analysis for disease or physiological status play the effect of vertical contrast.

Can, for the unaffected individuality that does not there is specific phenotype (thering is different ages, ethnic background and sex), by the vesica amount of measuring in unaffected individuality, set up reference point.For example, the reference point of reference group can be as detect the baseline of one or more vesica colonies in test subject.If the sample from the experimenter has the level similar to described reference point or value, described experimenter can be accredited as and not suffer from described disease, or the low possibility of described disease occurs.

Perhaps, can, for the individuality with particular phenotype, by the amount of measuring one or more vesica colonies in thering is the individuality of described phenotype, set up reference point or level.In addition, can be for the index of specific phenotype establishment value.For example, different disease stages can have different values, such as deriving from the individuality with various disease stage.Experimenter's value and described index can be compared, and can determine diagnosis or the prognosis of described disease, for example stage of disease or process.In other embodiments, created the index of value for curative effect.For example, can set up the vesica level of the individuality of suffering from specified disease, and to indicate which type for the treatment of for this individuality be effective.The value that described level can compare for the value created with the experimenter, and can processing or treatment for this individual selection, for example, by may be that treatment is respondent or non-respondent from the described experimenter of described horizontal forecast.

In some embodiments, can separate or detect vesica with the antigen of the biomarker of target particular cancers specifically, thereby determine reference point for the individuality that not affected by described particular cancers.As limiting examples, can use with for uninfluenced individuality described constructed inspection suffer from the individuality of different steps colorectal carcinoma and non-cancer polyp, and can measure each the group the circulating vesica level.In some embodiments, described level is defined as coming from the mean value ± standard deviation of at least two independent experiments to repeat at least in triplicate.Can use statistical test to carry out comparison between these groups to determine the statistical significance of distinguishing viewed biomarker.In some embodiments, statistical significance is determined in the operation parameter statistical test.Described parametric statistical test can include but not limited to, fractional factorial design, variance analysis (ANOVA), t check, method of least squares, Pearson are relevant, simple linear regression, non-linear regression, multivariate linear regression or Multiple Non Linear Regression.Perhaps, described parametric statistical test can comprise one-way analysis of variance, two-way analysis of variance or replicate measurement variance analysis.In other embodiments, use the nonparametric statistics check to determine statistical significance.The example includes but not limited to, Wilcoxen signed rank test (Wilcoxon signed-rank test), graceful-Whitney check (Mann-Whitney test), Kruskal-Wallis check, friedman's test (Friedman test), Spearman Rank correlation coefficient (Spearman ranked order correlation coefficient), Kendall Tau analysis and non parametric regression check.In some embodiments, statistical significance is determined under the p value lower than 0.05,0.01,0.005,0.001,0.0005 or 0.0001.Also can proofread and correct the p value for multiple comparisons, for example use that Bang Fulangni proofreaies and correct (Bonferroni correction), it is improved one's methods or other technology known to those of skill in the art, as Hochberg proofreaies and correct, Holm-Bonferroni proofreaies and correct,

correction, Dunnett ' s proofread and correct or Tukey ' s multiple comparisons.In some embodiments, for carrying out comparing after the check from the biomarker of each colony, carried out Tukey ' s and proofreaied and correct after ANOVA.

Can also set up reference point with the recurrence monitoring for disease (or deterioration stage of MS), be used for the treatment of reaction monitoring or for predicated response person/non-respondent's state.

In some embodiments, use artificial vesica (this paper also is called synthetic vesica) to measure the reference point for the microRNA available from vesica.Method for the preparation of artificial vesica is known to those skilled in the art, as used liposome.Can use the method be disclosed in US20060222654 and US4448765 to prepare artificial vesica, it is incorporated to this paper in full by reference.Can use markers with known to build artificial vesica is beneficial to catch and/or detect.In some embodiments, before processing, artificial vesica is mixed in body sample.Can during processing, follow the tracks of the level (for example, use and filter or other separation method disclosed herein) of complete synthetic vesica so that the contrast of initial sample to the vesica amount of processing sample to be provided.Similarly, during artificial vesica can mix sample before or after any treatment step.In some embodiments, artificial vesica is used for the equipment of vesica separation and detection for calibration.

Artificial vesica can and be used the feasibility of contrast with test analysis (such as the analysis based on pearl) through preparation.Described artificial vesica and described pearl and with detect antibodies.Therefore, aminoacid sequence/conformation that described artificial vesica comprises each antibodies.Described artificial vesica can comprise protein purification or the synthetic peptide sequence of antibodies.Described artificial vesica can be the pearl that can make biomolecules be attached thereto, as polystyrene bead.If described pearl has available carboxylic group, described protein or peptide can be incorporated into via available amino group described pearl, such as using the carbodiimide coupling.

In another embodiment, described artificial vesica can be the polystyrene bead that is coated with avidin, and vitamin H is placed in selected protein or peptide when synthetic or by vitamin H-maleimide chemical reaction.The proteins/peptides be placed on pearl can be mixed under the peculiar ratio of the application that will use described artificial vesica, and subsequently with described pearl coupling.These artificial vesicas can play a role as the connection of catching pearl and detect between antibody subsequently, provide therefrom contrast working properly in order to the component that shows described analysis.

Described value can be for quantitative value or is worth qualitatively.Described value can be directly the measuring of vesica level (for example mass/volume), or is indirect measurement, such as the amount of specific biological mark.Described value can be for quantitative, for example numerical value.In other embodiments, described value, for qualitatively, does not for example have the vesica of vesica, low-level vesica, medium level, high-caliber vesica or its modification.

Reference point can be stored in database, and can be for example, for example, according to the level of microRNA or the amount of amount (total amount of microRNA) or microRNA special group (the specific microRNA of cell source or from the microRNA of the vesica with particular organisms marking) and as diagnosis, prognosis, treatment diagnosis, disease classification, diseases monitoring, treatment monitoring, respondent/non-respondent's status predication with reference to for disease or situation.In an illustrative example, the method for determining cancer diagnosis is taken in.Be assessed and be stored in database from the microRNA of suffering from or do not suffer from the reference subject of described cancer.Described reference subject provides the described cancer of indication or another state, as the biological marking of state of health.Subsequent analysis compares from the sample of test subject and by the biological marking in the biological marking of microRNA and described database.If described experimenter's the biological marking is more closely relevant to the reference point of indication cancer, can make the diagnosis of cancer.On the contrary, if described experimenter's the biological marking is more closely relevant to the reference point of indication state of health, can determine that described experimenter does not suffer from described disease.Those skilled in the art should understand, and this example is nonrestrictive and can be expanded for assessment of other phenotype, as Other diseases, prognosis, treatment diagnosis, disease classification, diseases monitoring, treatment monitoring or respondent/non-respondent's status predication etc.

Can be identified for characterizing by detecting microRNA and/or vesica the biological marking of phenotype.Can in vesica, assess microRNA.Perhaps, in the situation that microRNA is not separated from vesica the microRNA in sample and vesica analyzed is characterized to described phenotype.Exist many analytical technologies to can be used for assessing vesica.In some embodiments, can identify according to mass spectrometry, flow cytometry, immunocytochemical stain, Western trace, electrophoresis, chromatogram or x radiocrystallography for methods known in the art the level of vesica.For example, can be according to Clayton etc., Journal of Immunological Methods 2001; Described in 163-174, use flow cytometry to be identified and quantitative measurment vesica, described document is incorporated herein by reference in full.Can determine with wedding agent the level of vesica according to mentioned above.For example, the wedding agent of vesica can be labeled, then certification mark thing for determining the amount of sample vesica.As described above, described wedding agent can be incorporated into substrate, such as array or particle.Perhaps, direct mark vesica.

Electrophoretic tag or eTag can be for measuring the amount of vesica.ETag is the little fluorescence molecule be connected with nucleic acid or antibody, and is designed to respectively and a kind of specific nucleic acid sequence or protein bound.At eTag, after its target is combined, use the combining eTag of enzyme to cut from target.In the signal produced by the eTag (being called " report thing ") discharged and sample, the amount of target nucleic acid or protein is proportional.Can identify eTag report thing by capillary electrophoresis.The charge-to-mass ratio (that is, its electric charge is divided by its molecular weight) of the uniqueness of each eTag report thing makes it show with specific peak on the capillary electrophoresis reading.Thus, by the particular organisms mark in vesica by the eTag target, can measure amount or the level of vesica.

Can for example, by heterogeneous vesica colony (the total vesica colony in sample), determine the vesica level.Perhaps, by the vesica colony of homogeneous or in fact the vesica colony of homogeneous determine the vesica level, such as the level of specific cells source vesica, as the vesica from prostate cancer cell.In other embodiment again, measure the level of for example, vesica with specific biomarker or biomarker combinations (prostate cancer being had to specific biomarker).Measure the vesica level and can be combined with the biomarker of definite vesica or biomarker combinations enforcement.Perhaps, can be before the biomarker of determining vesica or biomarker combinations or measure afterwards the amount of vesica.

Mensuration to the vesica amount can be analyzed in multiple mode.For example, can measure for example, amount more than a kind of vesica colony (the different cell source specificity vesicas that there is different biomarkers or biomarker combinations), as disclosed herein those.

Usually use the performance of statistics means assessment diagnosis or related check.Can assess by measuring sensitivity, specificity and calculation of correlation the performance of described sign.For example, but the level of evaluating objects microRNA is to characterize phenotype, as detected disease.Measured described analysis for the sensitivity and the specificity that detect described disease.

True positives is to have feature (as disease or imbalance) correctly to be differentiated the experimenter for feature as described in having.False positive is accredited as by described test the experimenter who has described feature and do not have described feature undeservedly.True negative correctly is accredited as the experimenter who does not have described feature without described feature by described test.False negative is have described feature and be accredited as undeservedly the people without this feature by described test.The ability that these classifications are distinguished in described test provides the tolerance of test performance.

The specificity of test is defined as the true negative number divided by actual negative (that is, true negative and false positive sum) number.Specificity is that how many experimenters are correctly differentiated negative measuring.It is all actual negative that 100% specificity means that described test identifies, and for example, whole healthy personages should be identified as health.Lower specificity shows that more feminine gender can be confirmed as the positive.

The sensitivity of test is defined as the true positives number divided by actual positive (that is, true positives and false negative sum) number.Specificity is that how many experimenters are correctly differentiated positive measuring.100% sensitivity mean described test identify whole actual positive-for example, whole ill personages should be identified as ill.Lower sensitivity shows that the more positive can be defined as feminine gender mistakenly.

The accuracy of test is defined as the number of true positives and true negative divided by all true positives and false positive and all true negative and false negative sum.It provides one sensitivity and specificity are measured to the numerical value combined.

Determine sensitivity, specificity and accuracy under specific identification threshold value.For example, the common threshold that prostate cancer (PCa) detects is the prostate specific antigen (PSA) of 4ng/mL in serum.The PSA level that is equal to or higher than described threshold value be considered to for PCa be positive and under any level be considered to negative.Along with changes of threshold, sensitivity and specificity also change.For example, along with the threshold value that detects cancer improves, specificity improves, and this is that it has caused false positive still less because more be difficult to take the experimenter as the positive.Meanwhile, sensitivity will reduce.Experimenter's performance curve (ROC curve) is along with changes of threshold, the schematic diagram of the True Positive Rate of Dual classification system (being sensitivity) to false positive rate (that is, 1-specificity).Described ROC curve display sensitivity and specificity how along with changes of threshold, to change.The area under curve of ROC curve (AUC) provides the total count value of the test performance of indication on the gamut of threshold value.Described AUC equals the positive sample that classification will select at random and is classified as the probability higher than the random cloudy shape sample of selecting.0.5 AUC show that described test has the probability of 50% suitable classification, it is equivalent to without identification power (toss a coin also have 50% correct classification probability).1.0 AUC mean that described test is suitably to whole experimenter's classifications (classification).AUC is equal to the Wilcoxon rank test.

Can for example, for the sensitivity with at least 50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69 or 70% (, with at least 71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86 or 87% sensitivity), characterize phenotype according to the biological marking of the present invention.In some embodiments, described phenotype is characterized with at least 87.1,87.2,87.3,87.4,87.5,87.6,87.7,87.8,87.9,88.0 or 89% sensitivity, for example at least 90% sensitivity.Described phenotype can be characterized with at least 91,92,93,94,95,96,97,98,99 or 100% sensitivity.

Can be for at least 50 according to the biological marking of the present invention, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96 or 97% specificity is (for example, with at least 97.1, 97.2, 97.3, 97.4, 97.5, 97.6, 97.7, 97.8, 97.8, 97.9, 98.0, 98.1, 98.2, 98.3, 98.4, 98.5, 98.6, 98.7, 98.8, 98.9, 99.0, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 100% specificity) characterize experimenter's phenotype.

Can be for the sensitivity and at least 60 with at least 50%, 65,70,75,80,85,90,95,99 or 100% specificity according to the biological marking of the present invention; At least 55% sensitivity and at least 60,65,70,75,80,85,90,95,99 or 100% specificity; At least 60% sensitivity and at least 60,65,70,75,80,85,90,95,99 or 100% specificity; At least 65% sensitivity and at least 60,65,70,75,80,85,90,95,99 or 100% specificity; At least 70% sensitivity and at least 60,65,70,75,80,85,90,95,99 or 100% specificity; At least 80% sensitivity and at least 60,65,70,75,80,85,90,95,99 or 100% specificity; At least 85% sensitivity and at least 60,65,70,75,80,85,90,95,99 or 100% specificity; At least 86% sensitivity and at least 60,65,70,75,80,85,90,95,99 or 100% specificity; At least 87% sensitivity and at least 60,65,70,75,80,85,90,95,99 or 100% specificity; At least 88% sensitivity and at least 60,65,70,75,80,85,90,95,99 or 100% specificity; At least 89% sensitivity and at least 60,65,70,75,80,85,90,95,99 or 100% specificity; At least 90% sensitivity and at least 60,65,70,75,80,85,90,95,99 or 100% specificity; At least 91% sensitivity and at least 60,65,70,75,80,85,90,95,99 or 100% specificity; At least 92% sensitivity and at least 60,65,70,75,80,85,90,95,99 or 100% specificity; At least 93% sensitivity and at least 60,65,70,75,80,85,90,95,99 or 100% specificity; At least 94% sensitivity and at least 60,65,70,75,80,85,90,95,99 or 100% specificity; At least 95% sensitivity and at least 60,65,70,75,80,85,90,95,99 or 100% specificity; At least 96% sensitivity and at least 60,65,70,75,80,85,90,95,99 or 100% specificity; At least 97% sensitivity and at least 60,65,70,75,80,85,90,95,99 or 100% specificity; At least 98% sensitivity and at least 60,65,70,75,80,85,90,95,99 or 100% specificity; At least 99% sensitivity and at least 60,65,70,75,80,85,90,95,99 or 100% specificity; Or be essentially the phenotype (for example, based on microRNA level or further feature) that 100% sensitivity and at least 60,65,70,75,80,85,90,95,99 or 100% specificity characterize the experimenter.

Can be for at least 60 according to the biological marking of the present invention, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96 or 97% accuracy is (for example, with at least 97.1, 97.2, 97.3, 97.4, 97.5, 97.6, 97.7, 97.8, 97.8, 97.9, 98.0, 98.1, 98.2, 98.3, 98.4, 98.5, 98.6, 98.7, 98.8, 98.9, 99.0, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 100% accuracy) characterize experimenter's phenotype.

In some embodiments, can be for at least 0.60 according to the biological marking of the present invention, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96 or 0.97 AUC is (for example, with at least 0.971, 0.972, 0.973, 0.974, 0.975, 0.976, 0.977, 0.978, 0.978, 0.979, 0.980, 0.981, 0.982, 0.983, 0.984, 0.985, 0.986, 0.987, 0.988, 0.989, 0.99, 0.991, 0.992, 0.993, 0.994, 0.995, 0.996, 0.997, 0.998, 0.999 or 1.00 AUC) characterize experimenter's phenotype.

In addition, can be identified for measuring with at least 50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98 or 99% degree of confidence the confidence level of specificity, sensitivity, accuracy or AUC.

Measuring of other correlated performance comprises positive and negative likelihood ratio [positive LR=sensitivity/(1-specificity); Negative LR=(1-sensitivity)/specificity].These measure the test performance that also can be used for estimating the method according to this invention.

Classification

The biological marking according to the present invention can be used for sample classification.The identification analytical technology it is known to those skilled in the art that.For example, can classify sample as or be predicted as respondent or the non-respondent for the given treatment for given disease or imbalance.Multiple statistical discriminant technique it is known to those skilled in the art that.In the supervised learning method, used the statistical classification methods analyst from the sample sets of two or more groups.Can find to can be used for setting up the biomarker of the sorter of distinguishing described two or more groups.Thereby can analyze subsequently new sample can be associated described fresh sample described sorter with in described two or more groups one group.Supervised classifier commonly used includes but not limited to neural network (multilayer perceptron), SVMs, k next-door neighbour algorithm, gauss hybrid models, Gaussian processes, naive Bayesian method (naive Bayes), decision tree and RBF (RBF) sorter.Linear classification comprises expense snow line differentiation (Fisher ' s linear discriminant), logistic regression, Naive Bayes Classifier, perceptron and SVMs (SVM).Comprise quadratic classifier, k next-door neighbour algorithm, strengthen algorithm, decision tree, random forest method, neural network, pattern recognition, Bayesian network (Bayesian networks) and hidden Markov model (Hidden Markov models) for other sorter of the present invention.The technician should be appreciated that, these and other sorter (comprising the wherein improvement of arbitrary classification device) is conceived within the scope of the present invention.

Use the classification that measure of supervision carries out usually to implement by following method:

For the given problem that solves supervised learning (as, study identification hand-written script), must consider various different steps:

1. acquisition training set.This can comprise, for example, and from suffering from or the experimenter of do not take a disease disease or imbalance, knownly responding or unresponsive experimenter, its disease have development or without experimenter's of development etc. sample for treatment.Described training sample is for " training " described sorter.

2. the input " feature " of determining learning function (learned function) means.The accuracy of described learning function depends on how to mean input object.Generally input object is transformed into to proper vector, it comprises a plurality of features for described object factory.Due to the cause of dimension disaster (curse of dimensionality), the number of feature should be not excessive; But should be even as big as the output that calculates to a nicety.Described feature can comprise one group of biomarker, such as the biomarker that is derived from vesica described herein.

3. determine structure and the corresponding learning algorithm of learning function.Select learning algorithm, for example, artificial neural network, decision tree, Bayes classifier or SVMs.Described learning algorithm is for setting up this sorter.

4. set up described sorter.The training set that the learning algorithm operation is collected.Can by optimize the Asia of described training set collection (being called the checking collection) performance or pass through the parameter that cross validation is adjusted described learning algorithm.After parameter adjustment and study, can be in the upper performance of measuring described algorithm of the test set (it separates with described training set) of primary sample.

Once determine as mentioned above sorter, it can be used for sample classification, for example, the experimenter's who is analyzed with method of the present invention sample.For example, can use suffer from and the reference subject of disease of not taking a disease in the data of target microRNA level set up sorter as described training set and test set.MicroRNA level seen in sample from test subject is assessed and described sorter suffers from or do not suffer from described disease for described experimenter is categorized as.The another example of lifting, can use found for specified disease respond or unresponsive reference subject in the data of target vesica biomarker level set up sorter as described training set and test set.Vesica biomarker level seen in sample from test subject is assessed and described sorter is suffered from or do not suffer from described disease for described experimenter is categorized as.

Also can be by the unsupervised learning method for the present invention.Clustering procedure is a kind of unsupervised learning method, and wherein clustering algorithm is in the situation that non-applying marking is carried out association by series of samples.The most similarly sample is classified into " group ".Can be categorized in the group by new sample and sort out with its tightst other associated member thus.The known a large amount of clustering algorithms of those skilled in the art can be used for the present invention, such as the hierarchical clustering method.

The biological marking

Obtain the biological marking according to the present invention by assessment vesica colony, comprise vesica associated biomolecule mark and/or the circulating biological mark of surface and useful load, comprise microRNA and protein.The biological marking that is derived from the experimenter can be used for characterizing described experimenter's phenotype.The biological marking can further comprise the level of the biomarker that one or more are other, for example, and circulating biological mark or the biomarker relevant to the target vesica.The biological marking of target vesica can comprise specific antigen or the biomarker be present on described vesica.The described biological marking also can comprise one or more antigens or biomarker, and it is carried as the useful load in vesica, comprises the microRNA of testing.The described biological marking can comprise one or more antigens of being present on vesica (it has one or more biomarkers that detect in vesica) or the combination of biomarker.The described biological marking can further comprise the out of Memory about vesica except biomarker.These information can comprise in vesica size, circulating half-life, metabolic half life and body or external given activity.The described biological marking can comprise described biomarker or for setting up the further feature of sorter.

In some embodiments, directly in biological sample, detect microRNA.For example, the RNA in body fluid can be used commercially available test kit to be separated, such as mirVana test kit (Applied Biosystems/Ambion, Austin, TX), MagMAX ^tMrNA separating kit (Applied Biosystems/Ambion, Austin, TX) and QIAzol Lysis Reagent and RNeasy Midi test kit (Qiagen Inc., Valencia CA).Can be according to hereinafter describing the particular types that uses array or round pcr to measure microRNA.

In some embodiments, the microRNA useful load of vesica is assessed to characterize phenotype.Can purifying or concentrated described vesica, then definite described biological marking.For example, separable cell source specificity vesica measure its biological marking.Perhaps, can be in the situation that without purifying in advance or concentrated directly from the biological marking of sample determination vesica.The biological marking of the present invention can be diagnosed for diagnosis, prognosis or the treatment of determining disease or situation, or similar measuring as herein described.The biological marking can also be for the stage of definite treatment effect, disease or situation or process or respondent/non-respondent's state of disease or situation.In addition, the biological marking can for example, for definite physiological status, gestation.

Can in vesica or the vesica feature of vesica itself assessed to determine the biological marking.Described feature can be for stage or the process of diagnosis, detection or definite disease, the treatment meaning of disease or situation, or for characterizing physiological status.Timeliness assessment, circulating vesica transformation period, the metabolic half life of vesica or the activity of vesica that these features include but not limited to the size of the level of vesica or amount, vesica, the vesica transformation period is changed.

One or more protein or peptide (for example providing protein imprinted), nucleic acid (for example,, as the described RNA marking or the DNA marking), lipid (for example lipid marking) or their combination are provided the biomarker that can comprise in the biological marking.In some embodiments, the described biological marking (for example can also comprise the type of the medicine that is present in vesica or drug metabolite or amount, provide the medicine marking), because these medicines can by described biological sample available from the experimenter take in, this has produced the vesica of the metabolite that carries described medicine or described medicine.

The biological marking also can comprise one or more biomarkers expression level, existence, do not exist, sudden change, varient, number of copies variation, brachymemma, repetition, modification or molecule association.Genetic variant or nucleotide variants refer to variation or the change at the specific gene seat of gene or cDNA sequence, and it includes but not limited to, nucleotide base disappearance, insertion, inversion and displacement in coding and non-coding region.Disappearance can be a part or the disappearance in a zone or the disappearance of complete genome sequence of the nucleotide sequence of the disappearance of mononucleotide base, described gene.Insertion can be the insertion of one or more nucleotide bases.Described genetic variant can be present in untranslated zone, exon, intron or the exon/intron connecting zone of transcriptional control zone, mRNA.Described genetic variant can cause or not cause the genetic transcription thing splicing form of terminator codon, frameshit, aminoacid deletion, change or the aminoacid sequence of change.

In embodiments, the biological nucleic acid mark that comprises the nucleic acid useful load in vesica carries out the assessment of nucleotide variants.Described biological nucleic acid mark can comprise one or more RNA materials, for example, and mRNA, miRNA, snoRNA, snRNA, rRNA, tRNA, siRNA, hnRNA, shRNA or its combination.Similarly, can assess the DNA useful load to form the DNA marking.

The RNA marking or the DNA marking also can comprise the outer genetic modification of sudden change, or are present in RNA in vesica or the genetic variant analysis of DNA.Outer genetic modification comprises the DNA methylation pattern.For example, referring to Lesche R. and Eckhardt F., DNA methylation markers:a versatile diagnostic tool for routine clinical use.Curr Opin Mol Ther.2007 June; 9 (3): 222-30, it is incorporated to this paper in full by reference.Therefore, biomarker can be the methylation state of DNA fragmentation.

The biological marking can comprise one or more miRNA markings that combine with one or more other markings (including but not limited to the mRNA marking, the DNA marking, protein imprinted, the peptide marking, the antigen marking or their any combination).For example, the described biological marking can comprise one or more miRNA biomarkers and one or more DNA biomarkers, one or more mRNA biomarkers, one or more snoRNA biomarkers, one or more protein biomarkers, one or more peptide biomarkers, one or more antigen biomarkers, one or more antigen biomarkers, one or more lipid biomarkers or their arbitrary combination.

The biological marking can comprise the combination ability of one or more wedding agents (for example in conjunction with) of one or more antigens or wedding agent, such as list in respectively in Fig. 1 and Fig. 2 or its place of this paper is described those.The described biological marking can further comprise one or more other biomarker, for example, such as but not limited to miRNA, DNA (single stranded DNA, complementary DNA or noncoding DNA) or mRNA.The biological marking of vesica can comprise one or more antigens (for example as shown in Figure 1), one or more wedding agents (for example as shown in Figure 2) and for example, for the combination of one or more biomarkers (as shown in Fig. 3-60) of situation or disease.The described biological marking can comprise one or more biomarkers (for example miRNA) and cancer cells is had to specific one or more antigens (for example, as shown in Figure 1).

In some embodiments, vesica for present method has the biological marking that is specific to described cell source, and for obtaining the specific diagnosis of disease specific or biological aspect, prognosis or the treatment associated biomolecule marking (it is representational for cell source).In other embodiments, vesica has for given disease or the specific biological marking of physiological situation, it is different from for diagnosis, prognosis, by stages, the biological marking of cell source that characterizes for the treatment of relativity determination or physiological status.The biological marking also can comprise the combination of cell source specificity and nonspecific vesica.

The biological marking can be for assessment of Case definition, the monitoring of for example existence of disease, staging, disease surveillance, disease classification or detection, the transfer of disease, recurrence or progress.The biological marking also can, clinically for being related to the decision-making of the pattern for the treatment of, comprise Results.The biological marking can comprise whether implementing operation further clinically for making the treatment decision-making, or should use which kind for the treatment of standard (for example preoperative or postoperative) together with operation.As illustrative example, the biological marking of microRNA (miRNA) of aggressive form of indication cancer may need more radical surgical measure and/or more radical treatment plan to treat described patient.

The biological marking can be used for the treatment of relevant diagnosis, thereby the test that can be used for diagnosing the illness or select correct treatment plan is provided, and the treatment diagnosis for example is provided.Treatment diagnosis comprises diagnostic test, and it provides the therapy that affects morbid state or the ability for the treatment of.The treatment diagnostic test is to provide respectively diagnosis or the similar mode of prognosis that the treatment diagnosis is provided with diagnosis or prognosis test.The treatment dependence test of any desired form has been contained in the treatment diagnosis that this paper is used, and it comprises prospective medicine, personalized medicine, synthetic medicine, pharmacodiagnosis (pharmacodiagnostics) and Dx/Rx partner (Dx/Rx partnering).The treatment dependence test can for the drug reaction of predicting and evaluating individual subjects, that is, provide personalized medical treatment.Predict drug response can determine whether the experimenter is possible respondent or the non-respondent of possibility of candidate therapeutic agent, for example, and before described experimenter is exposed to described treatment or is additionally processed with described treatment.The assessment drug reaction can be monitored the reaction to medicine, for example, and monitoring experimenter's improvement or the shortage of improvement in one period after begin treatment.The treatment dependence test can be used for for the Therapeutic selection experimenter, and described experimenter may benefit from especially described treatment or the early stage or objective indication with treatment effect likely is provided especially in individual subjects.Therefore, the biological marking disclosed herein can be indicated and should be changed treatment to select treatment more likely, has avoided thus delaying the great cost of useful treatment and has avoided using Financial cost and the ailing cost of invalid medicine.

In addition, the treatment dependent diagnostic can also be for the clinical diagnosis to various diseases or imbalance and management, and described disease or imbalance include but not limited to relative disease and autoimmunization relative disease in cardiovascular disorder, cancer, infectious diseases, Sepsis, sacred disease, central nervous system relative disease, blood vessel.The treatment dependent diagnostic also contributes to the prediction to drug toxicity, drug resistance or drug reaction.Can develop the treatment dependence test of arbitrarily suitable diagnostic test form, it includes but not limited to (for example) immunohistochemistry testing method, clinical chemistry, immunoassay, the technology based on cell, nucleic acid test or body formation method.The treatment dependence test may further include but be not limited to, and contributes to treat definite test, monitor therapy toxotest or to the test of the reaction for the treatment of.Therefore, the biological marking can be used for prediction or monitors the reaction of experimenter for treatment.After starting, remove or changing particular treatment, can measure the biological marking to the experimenter at different time points.

In some embodiments, make according to the amount of one or more components of the variation of the amount of one or more components of biomarker (that is, target microRNA, vesica and/or biomarker), particular organisms mark or the biomarker detected for described component mensuration or the prediction whether experimenter responds to treatment.In another embodiment, by different time points, measuring the situation that biomarker is monitored the experimenter.Determine situation process, disappear or recur.Also can on one section time-histories, measure the reaction to treatment.Therefore, the invention provides the method for state or other medical condition of monitoring of diseases in the experimenter, it comprises from the biological marking of the biological sample separation and detection from described experimenter, detect the particular organisms marking component total amount or detect the biological marking (such as the existence of biomarker, do not exist or expression level) of one or more components.The described biological marking can be used for monitoring the state of described disease or situation.

In some embodiments, the biological marking is for determining whether specified disease or situation have resistance for medicine.If the experimenter has resistance to medicine, the doctor without the time waste by valuable in this pharmacological agent.For obtaining the early stage checking to medicament selection or treatment plan, for the sample that derives from the experimenter, determine the biological marking.Whether the described biological marking has the biomarker relevant to drug resistance for assessment of the disease of particular subject.This determine can make the doctor that crucial time and patient's economic resources are put in effective treatment.

In addition, the biological marking can whether suffer from disease for assessment of the experimenter, whether in the risk in diseases or for assessment of stage or the process of disease.For example, whether the biological marking can suffer from prostate cancer (for example Figure 68,73) or colorectal carcinoma (for example Figure 69,74) for assessment of the experimenter.In addition, the biological marking can for example, for example, for determining the stage of disease or situation, colorectal carcinoma (Figure 71,72).

In addition, the amount of the amount of vesica (for example heterogeneous vesica colony) and one or more homogeneous vesica colonies (the vesica colony that for example has the identical biological marking) being measured can be for characterizing phenotype.For example, the total amount of vesica in sample (that is, acellular type specific) is measured and can be for characterizing phenotype to the determining of existence of one or more different cell source specificity vesicas.According to what hereinafter further describe, can according to normal subjects and the experimenter with target phenotype relatively come definite threshold or reference point or amount, and settle the standard according to determined threshold value or reference point.Different standards can be for characterizing phenotype.

A kind of standard can be based on the amount of heterogeneous vesica colony in sample.In one embodiment, general vesica mark (for example CD9, CD81 and CD63) can be for the amount of working sample vesica.And if can detect the described level of the expression level of CD9, CD81, CD63 or its combination higher than threshold level, meet described standard.In another embodiment, if the level of CD9, CD81, CD63 or its combination, lower than threshold value or reference point, meets described standard.Whether the amount that in another embodiment, described standard can be based on vesica is higher than threshold value or reference point.Another kind of standard can be based on thering is the particular organisms marking the amount of vesica.If there is the amount of vesica of the described particular organisms marking lower than threshold value or reference point, meet described standard.In another embodiment, if the amount of vesica with described particular organisms marking meets described standard higher than threshold value or reference point.The amount of the vesica that in addition, standard can also be based on being derived from particular cell types.If described amount, lower than threshold value or reference point, meets described standard.In another embodiment, if described amount, higher than threshold value, meets described standard.

In limiting examples, consider to measure the vesica from prostatic cell by detection of biological mark PCSA or PSCA, and if the level of the PCSA detected or PSCA higher than threshold level meet standard.Threshold value can be the level from identical mark in compared with control cells system or contrast experimenter's sample.Another kind of standard can be based on being derived from cancer cells or comprising one or more cancer specific biomarkers the amount of vesica.For example, can measure biomarker B7H3, EpCam or both, and if the level of the B7H3 detected and/or EpCam meets standard higher than threshold level or in pre-determined range.If described amount, below or above threshold value or reference point, meets described standard.Standard can be also the reliability of result, for example meets quality control method or value.The B7H3 detected in specimen and/or the amount of EpCam surpass the amount of these marks in control sample and can indicate cancer to be present in described specimen.

As described, can be by the analysis to multiple markers in addition in conjunction with assessment, whether meeting standard.In illustrative example, by detect in general vesica mark CD9, CD63 and CD81 one or more, comprise one or more prostatic epithelium mark and one or more cancer markers (as B7H3 and/or EpCam) of PCSA and PSMA, whether the biological marking is suffered to prostate cancer for assessment of the experimenter.Compare with the sample that contrasts individuality of not suffering from prostate cancer, shown the existence of prostate cancer in described experimenter from the higher level of mark in experimenter's sample.In some embodiments, assess described multiple markers in multiple mode.

The technician should understand, and these rules based on meeting described standard can be applicable to suitable biomarker arbitrarily.For example, described standard can be applicable to the vesica feature, such as amount, the microRNA of existence or the amount of other circulating biological mark of the vesica useful load biomarker of the vesica amount with particular organisms marking of the vesica amount existed, existence, existence, etc.Can measure the ratio of suitable biomarker.As illustrative example, described standard can be the ratio of ratio, a kind of circulating biological mark and the another kind of circulating biological mark of the ratio of ratio, vesica surface protein and the microRNA of vesica surface protein and another kind of vesica surface protein, a kind of vesica colony and another kind of vesica colony, etc.

Can be according to the phenotype that meets multiple useful standard and characterize the experimenter.In some embodiments, at least one standard is for each biomarker.In some embodiments, use at least 1,2,3,4,5,6,7,8,9,10,15,20,30,40,50,60,70,80,90 or at least 100 kinds of standards.For example, with regard to the sign of cancer, when described experimenter is diagnosed as while suffering from cancer, can use multiple different standard: 1) whether from the amount of microRNA in experimenter's sample higher than reference point; 2) whether the amount of the microRNA in cell type specificity vesica (that is, being derived from the vesica of specific tissue or organ) higher than reference point; Perhaps 3) whether there is the amount of microRNA in the vesica of one or more cancer specific biomarkers higher than reference point.If the amount of microRNA is lower than described reference point or identical with it, but rule like application class.Described method may further include quality control method, thereby provides result in the situation that described sample meets described quality control method for the experimenter.In some embodiments, if but meeting described standard quality control leaves a question open, and described experimenter reappraises.

In other embodiments, for the assess and determine of multiple biomarker single measuring, and described tolerance and reference point compare.As an example, can comprise to the test of prostate cancer the level that the level of PSA is multiplied by the miR-141 in blood sample.If the long-pending threshold value that surpasses of described level, meet described standard, this shows the existence of cancer.The another example of lifting, for the identical marker of multiple wedding agent portability of general vesica mark, as, identical fluorophore.Detected marker level and threshold value can be compared.

Beyond the multiple biomarker of same type, can be by standard application the biomarker in a plurality of types.For example, the level of one or more circulating biological marks (as RNA, DNA, peptide), vesica, sudden change etc. and reference can be compared.The heterogeneity of the biological marking can have different standards.As limiting examples, for the biological marking of diagnosing cancer can comprise a kind of miR material with reference to crossing of comparing, express and the vesica surface antigen with another with reference to the expression deficiency of comparing.

Can be by relatively amount, the structure of vesica or any out of Memory feature of vesica of vesica are determined the biological marking.Can use the structure of transmission electron microscopy (such as referring to Hansen etc., Journal of Biomechanics 31, Supplement 1:134-134 (1) (1998)) or scanning electron microscopy assessment vesica.Can using method and the multiple various combination of technology or the analysis of one or more vesicas is determined to experimenter's phenotype.

The biological marking can include but not limited to the existence of biomarker or do not exist, copy number, expression level or activity level.Other useful biological marking composition comprises the existence of sudden change (for example the impact transcribes or the sudden change of translation product activity, such as displacement, disappearance or insertion mutation), varient or the posttranslational modification of biomarker.The posttranslational modification of protein biomarker includes but not limited to the acidylate of described biomarker, acetylize, phosphorylation, ubiquitin, deacetylation, alkylation, methylate, amidation, biotinylation, γ-carboxylated, the glutamy amination, glycosylation, glycyl, hydroxylation, heme part covalently bound, iodate, isoprenylation, esterified, prenylation, the GPI grappling forms, myristoylation, farnesylation, spiceleaf acyl group spiceleaf acidylate, Nucleotide or derivatives thereof covalently bound, the ADP-ribosylation, flavine connects, oxidation, palmitoylation, Pegylation, phosphatidylinositols covalently bound, phosphopantetheine, how sialylated, Pyrrolidonecarboxylic acid forms, proline(Pro) racemization by prolyl isomerase, the aminoacid addition (for example arginyl) of tRNA mediation, sulfation, sulfate group adds on tyrosine or selenizing.

Method as herein described can also be for the identification of the biological marking relevant with disease, situation or physiological status.The described biological marking can also be for determining whether the experimenter suffers from cancer or whether in the risk of cancer occurs.Experimenter in risk in cancer occurs can comprise the experimenter of possibility susceptible or the experimenter with front symptom commitment disease.

Can also utilize the biological marking that the resolution of diagnosis or treatment is provided for Other diseases, described Other diseases includes but not limited to autoimmune disease, inflammatory bowel, alzheimer's disease, Parkinson's disease, multiple sclerosis, Sepsis, pancreatitis or listed any disease, situation or symptom in Fig. 3-58.

The described biological marking can also for example, for example, for identifying given pregnant state or disadvantageous pregnant result (mongolism being had to the specific miRNA marking), preeclampsia, premature labor, premature rupture of fetal membrane, intrauterine growth retardation or habitual abortion from peripheral blood, Cord blood or amniotic fluid.The described biological marking can also be used to indicate fetus, pre-implantation embryos or the neonatal health condition of mother, all etap.

The biological marking can be for the diagnosis of front symptom.In addition, the described biological marking can for detection of disease, the stage of determining disease or process, determine disease recurrence, confirm methods for the treatment of, determine effect or the evaluation individual physiological state relevant with age or environmental exposure of methods for the treatment of.

The biological marking of monitoring vesica also can be used for identifying experimenter's toxic exposure, and it includes but not limited to expose in early days or be exposed to situation unknown or unidentified toxic agent.By the particular theory of any mechanism of action, do not fettered, vesica can come off from damaging cells, and in this course the specific inclusion of cell is carried out to compartmentation, comprises film component and the cytoplasmic inclusion swallowed up.The cell that is exposed to toxic agents/chemical preparations may increase vesica come off discharge toxic agents or its metabolite, cause thus the vesica level improved.Therefore, monitoring vesica level, the biological marking of vesica or the individual reaction to the genotoxic potential agent of the two permission assessment.

By detecting one or more specific antigenss, wedding agent, biomarker or their any combination, can be by vesica and/or other biomarker of the present invention for the identification of drug-induced toxicity or the state of damaged organ.The variation of the biological marking of the level of vesica, vesica or the two can be exposed to for monitoring individual acute, chronic or occupational the situation of multiple toxic agents, and described toxic agents includes but not limited to medicine, microbiotic, technical chemistry goods, toxicity microbiotic metabolite, herbal medicine, daily-use chemical product and by natural formation or naturally synthesize the chemical substance by other organism generation.In addition, the biological marking can be for the identification of situation or disease, and it comprises the cancer of unknown origin, also referred to as the cancer (CUP) of not clear original site.

Thereby can be as previously described from biological sample, separate vesica and obtain heterogeneous vesica colony.Then, heterogeneous vesica colony is contacted with the substrate that is coated with specific-binding agent, described wedding agent is designed to get rid of or identifies the antigen-specific feature that given cell source is had to specific vesica colony.In addition, according to mentioned above, the biological marking of vesica can be associated with the cancerous state of cell.The compound that suppresses cancer in the experimenter can cause can be in time or therapeutic process by vesica is carried out to the variation that series of separate is monitored, for example variation of the biological marking of vesica.Can monitor vesica level with particular organisms marking or the variation of vesica level.

In one aspect, the present invention relates to biomarker discovery and the biological marking finds.In one embodiment, therapy is responded one or several experimenter (respondent) and can be subject to detecting its vesica to identical therapy unresponsive one or several experimenter (non-respondent).Can be detected to identify existing of one or more biomarkers, described biomarker comprises any biomarker as herein described.In one aspect, existence, quantity and the useful load of miR have been analyzed.The useful load of miR can be, for example, and surface or internal protein, nucleic acid, lipid or carbohydrate.

The respondent and in non-respondent, whether the existence of the biological marking not can be used for the treatment diagnosis.Can analyze the sample from the respondent for following one or more: the amount of vesica amount, unique vesica subgroup or kind, the biomarker in these vesicas, the biological marking of these vesicas, etc.In one case, vesica for the existence of one or more miRNA (such as miRNA 122, miR-548c-5p, miR-362-3p, miR-422a, miR-597, miR-429, miR-200a and/or miR-200b) and/or component analysis from respondent and non-respondent, such as microcapsule bubble or exosome.Between respondent and non-respondent, the difference of the biological marking can be used for the treatment diagnosis.In another embodiment, obtain vesica from the experimenter with disease or situation.Also never there is the experimenter of this disease or situation to obtain vesica.For unique biological marking, the vesica from two groups of experimenters is analyzed, the biological marking of described uniqueness is relevant but not relevant with the experimenter from another group to the whole experimenters in this group.These biological markings or biomarker can be used as the diagnosis whether existed for described situation or disease subsequently, or for described experimenter being ranged to the group (suffer from/do not take a disease disease, aggressive/Non-Invasive disease, respondent/non-respondent's group, etc.) of described group.

In further example, by suffering from I phase cancer, with the patient who suffers from the same cancer of II phase or III phase, vesica is analyzed.From the biomarker between each group patient's vesica or the difference of the biological marking (for example identified, the raising expression that may there is one or more genes or miR from the vesica of III phase cancer), the biological marking or the biomarker of distinguishing the disease different steps have been identified therefrom.Subsequently can be by this biology marking for the patient who suffers from described disease be carried out to prognosis.

In some cases, by for some time (for example, every day, weekly, per month or annual) inner analysis determines the biological marking from experimenter's vesica.Therefore, respondent and non-respondent or the patient in I phase and II/III phase in time (for example, per month) be subject to detecting its vesica.Can more described vesica in useful load or the physical properties at each time point place.Therefore temporal mode can form the biological marking that can be used for subsequently treating diagnosis, diagnosis, prognosis, disease classification, treatment monitoring, diseases monitoring or the person of responding/non-respondent's status predication.As unrestricted example, the amount that biomarker in vesica (as miR 122) improves with time-histories can be relevant to metastatic cancer, and this amount constant with time-histories to biomarker in vesica can be relevant with the non-metastatic cancer contrary.Time-histories is sustainable at least 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 6 weeks, 8 weeks, 2 months, 10 weeks, 12 weeks, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or at least 12 months.

The biological marking of the level of vesica, the level of vesica with particular organisms marking or vesica also can be for assessment of the treatment effect for situation.For example, the biological marking of the level of vesica, the level of vesica with fixed biological marking or vesica can be for assessment of the effect of cancer therapy, for example chemotherapy, radiotherapy or can be used for suppressing any other methods for the treatment of of the cancer in the experimenter.In addition, the biological marking can be analyzed material standed for or test compounds or the reagent (for example protein, peptide, plan peptide, class peptide, small molecules or other medicines) that the biological marking of vesica is had to the control effect to identify for screening.By described screening analyze that the compound identified can for example, for example suppress for () adjusting situation or disease, improvement, treatment or prevention situation or disease.

For example, the biological marking of vesica can derive from specific cancer is just being carried out to the patient of Successful treatment.Can not use the cancer patients's that identical medicine treated cell to be cultivated to deriving from, and obtain vesica with for determining the biological marking from described culture.Described cell can the use test compound treatment, and the biological marking that derives from the vesica of described culture can be compared with the biological marking that derives from the patient's who carries out Successful treatment vesica.The test compounds that can select to produce the biological marking similar to the patient's who just carries out Successful treatment the biological marking is with for further research.

In addition, the biological marking of vesica can also for example, for the impact of monitoring reagent (medical compounds) in clinical trial on the biological marking.In addition, the variation of the biological marking of level, vesica of monitoring vesica or the two also can be for assessment of the methods of the effect of test compounds, for example, for the test compounds of anticancer.

In addition, the biological marking of the level of vesica, vesica or the two can also be for determining particular treatment intervention (medicine or non-medicine) thus validity and for changing described intervention 1) reduce the risk that unfavorable result occurs, 2) strengthen the validity or 3 of intervening) differentiate the resistance state.Therefore, except the existence or occurrence risk of diagnosis or confirmation disease, situation or symptom, method and composition disclosed herein also provides the system of the treatment for optimizing the experimenter to suffering from described disease, situation or symptom.The biological marking that for example, can be tested and appraised vesica is determined the treatment relational approach (thereby it will be by diagnosing and treating and integrated the real-time treatment that improves the experimenter) for the treatment of disease, situation or symptom.

Level, the biological marking of vesica or the two the testing method of identifying vesica can be for the identification of the patients who is suitable for most particular treatment, and how medicine is played a role well feedback is provided, thus optimizing therapeutic regimen.For example, the hypertension of inducing in gestation and conditions associated in, treat relevant diagnosis and can monitor neatly in time the variation of important parameter (for example level of cytokine and/or somatomedin), thereby optimize treatment.

In the defined research of FDA, MDA, EMA, USDA and EMEA, with in the clinical trial situation of medicine, by the biological marking disclosed herein, definite relevant diagnosis for the treatment of can design, monitor for optimization Test effect and increase drug safety provides crucial information.For example, with regard to test design, treating relevant diagnosis can be for the establishment of determining (entering group/eliminating), homogeneity treatment group of patient's classification, patient's qualification and to the selection with patient's sample of coupling case-control cohort through optimization.Therefore, this treats the means that relevant diagnosis can provide patient's effect enrichment (patient efficacy enrichment), thus test is raised to required individual amount and is down to minimum.For example, with regard to effect, treat relevant diagnosis and can and assess effect standard for the monitor therapy diagnoses and treatment.Perhaps, with regard to security, treating relevant diagnosis can be for preventing ADR or avoiding malpractice and monitor the conformability to treatment plan.

In some embodiments, the invention provides the method for identifying for respondent and the non-respondent of the treatment of carrying out clinical trial, it is included in the patient who participates in described clinical trial and detects the biological marking that comprises microRNA, and identifies the biological markings different between respondent and non-respondent.In further embodiment, measure the described biological marking and use it for and predict that described experimenter should be respondent or non-respondent in medicine is just controlled the experimenter.Whether described prediction can just be controlled experimenter's the biological marking according to described medicine more closely associated with the clinical trial experimenter through being accredited as the respondent, predicts that therefrom described medicine just controls the experimenter and should be the respondent.On the contrary, more closely associated with the clinical trial experimenter through being accredited as non-respondent if described medicine is just controlled experimenter's the biological marking, the measurable described medicine of method of the present invention is just controlled the experimenter and be should be non-respondent.Therefore, described prediction can be used for potential response person and the non-respondent of described treatment are distinguished.In some embodiments, described prediction is used for instructing the course for the treatment of, as, treat the doctor by help and determine whether to use described medicine.In some embodiments, described prediction is for instructing the selection of the patient to participating in further clinical trial.In limiting examples, in testing in the II phase, the biological marking of predicated response person/non-respondent's state can be used for selecting the patient who carries out III phase test, has improved therefrom the possibility responded in III phase patient colony.The technician should understand, and described method can be through adjusting for the identification of the biological marking, thereby according to the standard except respondent/non-respondent's state, the experimenter is distinguished.In one embodiment, described standard is the treatment security.Therefore, according to above following described method to identify the experimenter who described treatment is likely there is or can not have undesirable condition.In limiting examples, in testing in the II phase, the biological marking of predictive of safety feature can be used for selecting the patient carry out III phase test, has improved thus the treatment security features in described III phase patient colony.

Therefore, the biological marking of described vesica level, vesica or both all can be used for monitoring drug effect, measure the reaction of given medicine or resistance or both, thereby have strengthened drug safety.For example, in colorectal carcinoma, vesica generally comes off and can separate from peripheral blood and for separating of one or more biomarkers from colon cancer cell, and as KRAS mRNA, it can be checked order to detect the KRAS sudden change subsequently.In the situation of mRNA biomarker, described mRNA can reverse transcription become cDNA and checked order (for example by Sanger check order, tetra-sodium order-checking, NextGen order-checking, RT-PCR analyze), thereby determine whether to exist the sudden change of giving medicine (for example Cetuximab or Victibix) resistance.In another embodiment, separate the vesica come off specifically from biological sample from lung carcinoma cell, and use it for the biomarker that separates lung cancer, for example EGFR mRNA.EGFR mRNA is processed into to cDNA order-checking, thereby determines whether to exist EGFR sudden change (it demonstrates the resistance of the particular medication for lung cancer or reaction).

One or more biological markings can be divided into groups, thereby make the information of the biological marking collection in obtained relevant particular group provide reasonable basis for carrying out clinical relevant decision-making, such as but not limited to diagnosis, prognosis or treatment management, for example treatment is selected.

The same with most of diagnostic marks, use the mark of the minimum number be enough to make correct medical judgment normally desirable.This has prevented in the treatment delay of products for further analysis and the inappropriate use of time and resource.

In addition, herein disclosed is the method for sample (for example serum and tissue biological storehouse) being implemented to retrospective analysis, to reach for example, by quantitative and qualitative analysis character (the biological marking of the vesica) clinical effectiveness with morbid state, disease stage, progress, prognosis aspect; Treatment effect or selection; The purpose that perhaps physiological situation is associated.In addition, method and composition disclosed herein is for for example, prospective analysis to sample (serum and/or the tissue collected by individuality in clinical trial), to reach the clinical effectiveness of the biological marking of the vesica of quantitative and qualitative analysis and morbid state, disease stage, progress, prognosis aspect; Treatment effect or selection; The purpose that perhaps physiological situation is associated.As used herein, the biological marking of vesica can be used for the specific vesica in identification of cell source.In addition, the biological marking can be determined according to the surface marker distribution of vesica or the inclusion of vesica.

For the biological marking that characterizes phenotype according to the present invention can comprise Multiple components (as, microRNA, vesica or other biomarker) or feature (as, vesica size or form).The described biological marking can comprise at least 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,25,30,40,50,75 or 100 kind of composition or feature.For example have, more than the biological marking of a kind of composition or feature (at least 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,25,30,40,50,75 or 100 kind of composition) and can provide higher sensitivity and/or specificity characterizing aspect phenotype.In some embodiments, with the situation of the less composition of assessment or feature, compare, assessment Multiple components or feature provide the sensitivity and/or the specificity that strengthen.On the other hand, use composition or the feature of the minimum number be enough to make correct medical judgment normally desirable.Mark still less can be avoided the statistics over-fitting of sorter and can prevent in the treatment delay of products for further analysis and the inappropriate use of time and resource.Therefore, method of the present invention comprises the optimal number of determining composition or feature.

The biological marking according to the present invention can be used for characterizing phenotype with above-mentioned sensitivity, specificity, accuracy or similar performance metric.The described biological marking also can be used for setting up sorter so that sample is classified as and belongs to a certain group, such as belonging to, suffers from or the group of the disease that do not take a disease, suffers from affecting conditions or do not suffer from group, respondent or the non-respondent's of affecting conditions group.In one embodiment, sorter is for determining whether the experimenter suffers from aggressive or Non-Invasive cancer.Under the exemplary cases of prostate cancer, this can assist the doctor to determine whether to described cancer is observed (that is, opening " observe and wait for " prescription) or implemented prostatectomy.In another embodiment, whether sorter likely responds to tamoxifen for definite patient with breast cancer or is reactionless, thereby auxiliary doctor determines whether to use tamoxifen or the described patient of another pharmacological agent.

Biomarker

Can comprise one or more biomarkers for the biological marking that characterizes phenotype.Described biomarker can be cycling markers, film Research of predicting markers or be present in vesica or the lip-deep composition of vesica.These biomarkers include but not limited to nucleic acid (such as RNA (mRNA, miRNA etc.) or DNA), protein, peptide, polypeptide, antigen, lipid, carbohydrate or proteoglycan.

The described biological marking can comprise the existence of biomarker (for example in Fig. 1,3-60 listed any or multiple biomarker) or do not exist, expression level, mutation status, hereditary variant state or any modification (for example outer genetic modification, posttranslational modification).Can by the expression level of biomarker with contrast or with reference to comparing, to determine that in sample, not enough (or raise or lower) expressed or expressed to crossing of biomarker.In some embodiments, described contrast or reference level comprise the amount that does not have or do not show the identical biomarker (for example miRNA) in experimenter's the control sample of situation or disease that derives from.In another embodiment, described contrast or reference level comprise that level in different biological situation (such as ill to disease state not) is subject to the level of house keeper's mark of minimum impact (if influential words).In another embodiment again, but described contrast or reference level comprise in same experimenter the level of identical mark in the sample gathered in different time points.This paper describes the contrast of other type.

The biological nucleic acid mark comprises various RNA or DNA material.For example, described biomarker can be mRNA, microRNA (miRNA), little nucleolar RNA (snoRNA), small nuclear rna (snRNA), ribosome-RNA(rRNA) (rRNA), heterogeneous nuclear RNA (hnRNA), ribosome-RNA(rRNA) (rRNA), siRNA, transfer RNA (tRNA) or shRNA.Described DNA can be double-stranded DNA, single stranded DNA, complementary DNA or noncoding DNA.MiRNA is short Yeast Nucleic Acid (RNA) molecule, and it on average is about 22 Nucleotide.MiRNA plays a role as transcribing rear conditioning agent, the complementary sequence in its 3 ' non-translational region in conjunction with target messenger RNA(mRNA) (mRNA) (3 ' UTR), and this can cause gene silencing.A kind of miRNA can act on 1000 kinds of mRNA.MiRNA brings into play multiple effect in negative regulation, for example, and transcript degraded and isolation, translation compacting, and can in positive regulation, play a role, for example, transcribe and translate activation.By affecting gene regulating, miRNA can affect many bioprocesss.Find different expression miRNA collection at different cell types with in tissue.

Further comprise peptide, polypeptide or protein for biomarker of the present invention, these terms are used in the text interchangeably, unless otherwise noted.In some embodiments, described protein biomarker comprise its decorating state, brachymemma, sudden change, expression level (such as, with reference level, compared express or express not enough) and/or posttranslational modification, for example mentioned above.In limiting examples, the biological marking of disease can comprise having the protein of modifying after certain translation, its in the sample with described disease association than more not general in associated sample with it.

The biological marking can comprise the biomarker (for example two kinds of different microRNA materials) of multiple same type, or one or more dissimilar biomarkers (for example mRNA, miRNA, protein, peptide, part and antigen).

One or more biological markings can comprise and are selected from least one biomarker listed in Fig. 1,3-60.The biological marking in specific cells source can comprise one or more biomarkers.The table of various diseases or situation specific biological mark has been described to have enumerated in Fig. 3-58, and wherein said biomarker can be obtained and be analyzed by vesica.In addition, described biomarker can also be adjusted element, TMPRSS2-ERG, PCA-3, PSA, EGFR, EGFRvIII, BRAF variant, MET, cKit, PDGFR, Wnt, beta-catenin, K-ras, H-ras, N-ras, Raf, N-myc, c-myc, IGFR, PI3K, Akt, BRCA1, BRCA2, PTEN, VEGFR-2, VEGFR-1, Tie-2, TEM-1, CD276, HER-2, HER-3 or HER-4 for CD24, Midkine (midkine), iron.In addition, described biomarker can also be annexin V, CD63, Rab-5b or caveolin or miRNA, for example let-7a, miR-15b, miR-16, miR-19b, miR-21, miR-26a, miR-27a, miR-92, miR-93, miR-320 or miR-20.In addition, described biomarker can also be disclosed any gene or its fragment in PCT publication number No.WO2009/100029, for example listed those in table 3-15 wherein.

Other biomarker that can be used for assessment in method and composition disclosed herein comprises and U.S. Patent No. 6329179 and 7,625,573, U.S. Patent Publication No. No.2002/106684,2004/005596,2005/0159378,2005/0064470,2006/116321,2007/0161004,2007/0077553,2007/104738,2007/0298118,2007/0172900,2008/0268429,2010/0062450,2007/0298118,2009/0220944 and 2010/0196426, U.S. Patent application No.12/524,432,12/524,398,12/524,462, Canadian Patent CA 2453198, and the open No.WO1994022018 of International PCT patent, WO2001036601, WO2003063690, WO2003044166, WO2003076603, WO2005121369, WO2005118806, WO/2005/078124, WO2007126386, WO2007088537, WO2007103572, WO2009019215, WO2009021322, WO2009036236, WO2009100029, WO2009015357, WO2009155505, WO2010/065968 and disclosed situation in WO 2010/070276 or the relevant biomarker of physiological status, each patent or application are incorporated to this paper in full by reference.The part that in these patents and application, disclosed biomarker (comprising vesica biomarker and microRNA) can be used as the marking for characterizing phenotype (such as the diagnosis that cancer or Other diseases are provided, prognosis or treatment diagnosis) is assessed.In addition, method disclosed herein and technology can be used for assessing biomarker, comprise vesica biomarker and microRNA.

Be used in another that assessed in method and composition disclosed herein and organize available biomarker and comprise the biomarker relevant to cancer diagnosis, prognosis or treatment diagnosis, as be disclosed in United States Patent (USP) 6,692,916,6,960,439,6,964,850,7,074,586; U.S. Patent application No.11/159,376,11/804,175,12/594,128,12/514,686,12/514,775,12/594,675,12/594,911,12/594,679,12/741,787,12/312,390; And in International PCT patent application No.PCT/US2009/049935, PCT/US2009/063138, PCT/US2010/000037; Each patent or application are incorporated to this paper in full by reference.Useful biomarker further comprises for inflammatory diseases at U.S. Patent application No.10/703,143 and US 10/701,391 in; For rheumatoid arthritis in 11/529,010; For multiple sclerosis in 11/454,553 and 11/827,892; For graft-rejection in 11/897,160; For lupus in 12/524,677; In PCT/US2009/048684 for osteoarthritis; For infectious diseases and Sepsis in 10/742,458; For Sepsis, in 12/520,675, describe; Each patent or application are incorporated to this paper in full by reference.The part that in these patents and application, disclosed biomarker (comprising microRNA) can be used as the marking for characterizing phenotype (such as the diagnosis that cancer or Other diseases are provided, prognosis or treatment diagnosis) is assessed.In addition, method disclosed herein and technology can be used for assessing biomarker, comprise vesica biomarker and microRNA.

Can be in method and composition disclosed herein for assessment of other biomarker again comprise the .Isolation and characterization of an RNA-proteolipid complex associated with the malignant state in humans with Wieczorek etc., Proc Natl Acad Sci U S is year May A.1985; 82 (10): 3455-9; The .Diagnostic and prognostic value of RNA-proteolipid in sera of patients with malignant disorders following therapy:first clinical evaluation of a novel tumor marker such as Wieczorek, Cancer Res.1987 December 1; 47 (23): 6407-12; Selective enrichment of tetraspan proteins on the internal vesicles of multivesicular endosomes and on exosomes secreted by human B-lymphocytes.J.Biol.Chem. (1998) 273:20121-27 such as Escola; The Binding of hepatitis C virus to CD81 Science such as Pileri, (1998) 282:938-41); The Detection of Tumor Messenger RNA in the Serum of Patients with Malignant Melanoma such as Kopreski, Clin.Cancer Res. (1999) 5:1961-1965; The Circulating Membrane Vesicles in Leukemic Blood such as Carr, Cancer Research, (1985) 45:5944-51; The Cytoplasmic CD24 expression in colorectal cancer independently correlates with shortened patient survival.Clinical Cancer Research such as Weichert, 2005,11:6574-81); MicroRNA gene expression deregulation in human breast cancer.Cancer Res (2005) 65:7065-70 such as Iorio; Tumour-derived exosomes and their role in cancer-associated T-cell signaling defects British J Cancer (2005) 92:305-11 such as Taylor; Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells Nature Cell Biol (2007) 9:654-59 such as Valadi; Pregnancy-associated exosomes and their modulation of T cell signaling J Immunol (2006) 176:1534-42 such as Taylor; The Purification such as Koga, characterization and biological significance of tumor-derived exosomes Anticancer Res (2005) 25:3703-08; Epithelial cell adhesion molecule (KSA) expression:pathobiology and its role as an independent predictor of survival in renal cell carcinoma Clin Cancer Res (2004) 10:2659-69 such as Seligson; Clayton etc. (Antigen-presenting cell exosomes are protected from complement-mediated lysis by expression of CD55 and CD59.Eur J Immunol (2003) 33:522-31); Cell Membrane Microparticles in Blood and Blood Products:Potentially Pathogenic Agents and Diagnostic Markers Trans Med Reviews (2006) 20:1-26 such as Simak; Proteomic analysis of microvesicles derived from human colorectal cancer cells J Proteome Res (2007) 6:4646-4655 such as Choi; Tumour-released exosomes and their implications in cancer immunity Cell Death Diff (2008) 15:80-88 such as Iero; Tumour-derived microvesicles carry several surface determinants and mRNA of tumour cells and transfer some of these determinants to monocytes Cencer Immunol Immunother (2006) 55:808-18 such as Baj-Krzyworzeka; B cell-derived exosomes can present allergen peptides and activate allergen-specific T cells to proliferate and produce TH2-like cytokines J Allergy Clin Immunol (2007) 120:1418-1424 such as Admyre; Identification and characterization of microvesicles secreted by 3T3-Ll adipocytes:redox-and hormone dependent induction of milk fat globule-epidermal growth factor 8-associated microvesicles Endocrinol (2007) 148:3850-3862 such as Aoki; Tumour-derived microvesicles carry several surface determinants and mRNA of tumour cells and transfer some of these determinants to monocytes Cencer Immunol Immunother (2006) 55:808-18 such as Baj-Krzyworzeka; Glioblastoma microvesicles transport RNA and proteins that promote rumour growth and provide diagnostic biomarkers Nature Cell Biol (2008) 10:1470-76 such as Skog; The Characterization of amplifiable such as El-Hefnawy, circulating RNA in plasma and its potential as a tool for cancer diagnostics Clin Chem (2004) 50:564-573; The .Proc Natl Acad Sci U S A such as Pisitkun, 2004; 101:13368-13373; The .Can urinary exosomes act as treatment response markers in Prostate Cancer such as Mitchell?, Journal of Translational Medicine 2009,7:4; The .Human Tumor-Derived Exosomes Selectively Impair Lymphocyte Responses to Interleukin-2 such as Clayton, Cancer Res 2007; 67:(15) .2007 August 1; Decay-accelerating factor (CD55) and membrane inhibitor of reactive lysis (CD59) the are released within exosomes during In vitro maturation of reticulocytes.Blood 91:2573-2580 (1998) such as Rabesandratana; The Production and characterization of clinical grade exosomes derived from dendritic cells.J Immunol Methods 270:211-226 (2002) such as Lamparski; CD24 is a marker of exosomes secreted into urine and amniotic fluid.Kidney Int ' the l 72:1095-1102 (2007) such as Keller; The Malignant ascties-derived exosomes of ovarian carcinoma patients contain CD24 and EpCAM.Gyn Oncol 107:563-571 (2007) such as Runz; The Circulating microparticles in normal pregnancy and preeclampsia placenta.29:73-77 (2008) such as Redman; The Cleavage of L such as Gutwein 1 in exosomes and apoptotic membrane vesicles released from ovarian carcinoma cells.Clin Cancer Res 11:2492-2501 (2005); The .CD24 is an independent prognostic marker of survival in nonsmall cell lung cancer patients such as Kristiansen, Brit J Cancer 88:231-236 (2003); Lim and Oh, The Role of CD24 in Various Human Epithelial Neoplasias, Pathol Res Pract 201:479-86 (2005); The .The Immunophenotype of Splenic Lymphoma with Villous Lymphocytes and its Relevance to the Differential Diagnosis With Other B-Cell Disorders such as Matutes, Blood 83:1558-1562 (1994); Pirruccello and Lang, Differential Expression of CD24-Related Epitopes in Mycosis Fungoides/Sezary Syndrome:A Potential Marker for Circulating Sezary Cells, disclosed situation or the relevant biomarker of physiological status in Blood 76:2343-2347 (1990).Disclosed biomarker in these publications (comprising vesica biomarker and microRNA) can be used as the part of the marking for characterizing phenotype (such as the diagnosis that cancer or Other diseases are provided, prognosis or treatment diagnosis) and is assessed.In addition, method disclosed herein and technology can be used for assessing biomarker, comprise vesica biomarker and microRNA.

Can be in method and composition disclosed herein for assessment of other biomarker again comprise and Rajendran etc., Proc Natl Acad Sci U S A 2006, 103:11172-11177, Taylor etc., Gynecol Oncol 2008, 110:13-21, Zhou etc., Kidney Int 2008, 74:613-621, Buning etc., the J Immunol 2008 such as Immunology 2008, Prado, 181:1519-1525, Vella etc. (2008) Vet Immunol Immunopathol 124 (3-4): 385-93, Gould etc. (2003) .Proc Natl Acad Sci U S A 100 (19): 10592-7, Fang etc. (2007) .PLoS Biol 5 (6): e158, Chen, B.J.and R.A.Lamb (2008) .Virology 372 (2): 221-32, Bhatnagar, S.and J.S.Schorey (2007) .J Biol Chem 282 (35): 25779-89, Bhatnagar etc. (2007) Blood 110 (9): 3234-44, Yuyama etc. (2008) .J Neurochem 105 (1): 217-24, Gomes etc. (2007) .Neurosci Lett 428 (1): 43-6, Nagahama etc. (2003) .Autoimmunity 36 (3): 125-31, Taylor, D.D., S.Akyol etc. (2006) .J Immunol 176 (3): 1534-42, Peche etc. (2006) .Am J Trans[lant 6 (7): 1541-50, Iero, M., M.Valenti etc. (2008) .Cell Death and Differentiation 15:80-88, Gesierich, S., I.Berezoversuskiy etc. (2006), Cancer Res 66 (14): 7083-94, Clayton, A., A.Turkes etc. (2004) .Faseb J 18 (9): 977-9, Skriner., K.Adolph etc. (2006) .Arthritis Rheum 54 (12): 3809-14, Brouwer, R., G.J.Pruijn etc. (2001) .Arthritis Res 3 (2): 102-6, Kim, S.H., N.Bianco etc. (2006) .Mol Ther 13 (2): 289-300, Evans, C.H., S.C.Ghivizzani etc. (2000) .Clin Orthop Relat Res (379 Suppl): S300-7, Zhang, H.G., C.Liu etc. (2006) .J Immunol 176 (12): 7385-93, Van Niel, G., J.Mallegol etc. (2004) .Gut 52:1690-1697, Fiasse, R. with O.Dewit (2007) .Expert Opinion on Therapeutic Patents 17 (12): disclosed situation or the relevant biomarker of physiological status in 1423-1441 (19).Disclosed biomarker in these publications (comprising vesica biomarker and microRNA) can be used as the part of the marking for characterizing phenotype (such as the diagnosis that cancer or Other diseases are provided, prognosis or treatment diagnosis) and is assessed.In addition, method disclosed herein and technology can be used for assessing biomarker, comprise vesica biomarker and microRNA.

Can be obtained and the biomarker analyzed is miRNA (miR), miRNA by vesica ^*nonsense (miR ^*) and other RNA (including but not limited to mRNA, preRNA, priRNA, hnRNA, snRNA, siRNA, shRNA).The miRNA biomarker not only comprises its miRNA and microRNA ^*nonsense, and comprise its precursor molecule: former microRNA (former miR) and front microRNA (front miR).The sequence of miRNA can obtain from disclosing available database, for example http://www.mirbase.org/, http://www.microma.org/ or any other available database.Described biomarker can also be nucleic acid molecule (as DNA), protein or peptide.Can measure the existence of described biomarker or do not exist, expression level, sudden change (for example transgenation, as disappearance, transposition, repetition, Nucleotide or amino-acid substitution etc.).Can also analyze any outer genetic regulation or the copy number variation of biomarker.

One or more biomarkers of being analyzed can be the indication of particular organization or cell derived, disease or physiological status.In addition, the existence of one or more biomarkers described herein, do not exist or expression level can be relevant to experimenter's phenotype (comprising disease, situation, prognosis or drug effect).The particular organisms mark hereinafter provided and the biological marking have formed the non-inclusive example of each disease, situation comparison, situation and/or physiological status.In addition, one or more biomarkers of assessing for phenotype can be the specific vesica of cell source.

For one or more miRNA that characterize phenotype, can be selected from the miRNA described in PCT publication number No.WO2009/036236.For example, in Table I-VI (Fig. 6-11), one or more listed miRNA can hide for characterizing adenocarcinoma of colon, colorectal carcinoma, prostate cancer, lung cancer, mammary cancer, b-cell lymphoma, carcinoma of the pancreas, diffuse large B CL cancer, CLL, bladder cancer, kidney, hypoxic tumor, hysteromyoma, ovarian cancer, hepatitis C virus related Hepatocellular Carcinoma, ALL, alzheimer's disease, myelofibrosis, myelofibrosis, polycythemia vera, thrombocytosis, HIV or HIV-I therein, as further described herein.

Can in vesica, detect one or more miRNA.Described one or more miRNA can be miR-223, miR-484, miR-191, miR-146a, miR-016, miR-026a, miR-222, miR-024, miR-126 and miR-32.In addition, can also in PBMC, detect one or more miRNA.Described one or more miRNA can be miR-223, miR-150, miR-146b, miR-016, miR-484, miR-146a, miR-191, miR-026a, miR-019b or miR-020a.Described one or more miRNA can be for characterizing specific disease or situation.For example, for the bladder cancer disease, can detect one or more miRNA, for example miR-223, miR-26b, miR-221, miR-103-1, miR-185, miR-23b, miR-203, miR-17-5p, miR-23a, miR-205 or their any combination.Described one or more miRNA can be raised or cross and express.

In some embodiments, described one or more miRNA are for characterizing hypoxic tumor.Described one or more miRNA can be miR-23, miR-24, miR-26, miR-27, miR-103, miR-107, miR-181, miR-210 or miR-213, and can raise.One or more miRNA can also be for characterizing hysteromyoma.For example, for one or more miRNA that characterize hysteromyoma, can be member, miR-21, miR-23b, miR-29b or the miR-197 of let-7 family.Described miRNA can be raised.

Can also characterize myelofibrosis, for example miR-190 (it can raise) by one or more miRNA; MiR-31, miR-150 and miR-95 (it can be lowered); Perhaps their any combination.In addition, can also characterize myelofibrosis, polycythemia vera or thrombocytosis by detecting one or more miRNA, such as but not limited to miR-34a, miR-342, miR-326, miR-105, miR-149, miR-147 or their any combination.Described one or more miRNA can lower.

Other example of the phenotype that can characterize by one or more biomarkers of assessment vesica further describes hereinafter.

Can use one or more biomarkers of probe in detecting.Probe can comprise chemical compound (including but not limited to medicine or labelled reagent), arborescence or their combination of oligonucleotide (for example DNA or RNA), fit, monoclonal antibody, polyclonal antibody, Fab, Fab ', single-chain antibody, synthetic antibody, class peptide, zDNA, peptide nucleic acid(PNA) (PNA), lock nucleic acid (LNA), lectin, synthetic or natural formation.Can carry out the described probe of direct-detection by for example direct mark, or carry out the described probe of indirect detection by for example labelled reagent.Described probe can optionally be identified biomarker.For example, as the probe of oligonucleotide, can optionally with the miRNA biomarker, hybridize.

In many aspects, the present invention is for being diagnosed, treated diagnosis, prognosis, disease classification, treatment monitoring or the person of responding/non-respondent's status predication to experimenter's disease or imbalance.The present invention includes the vesica from the experimenter is assessed, it comprises that assessment is present in the biomarker on described vesica and/or assesses the useful load in described vesica, such as protein, nucleic acid or other biomolecules.Can use vesica to be assessed and can be used for implementing method of the present invention to disease or the relevant any suitable biomarker of lacking of proper care.In addition, can use any suitable technical evaluation vesica as herein described.But the exemplary biomarker for specified disease that the method according to this invention is assessed comprises following:

Mammary cancer

Mammary cancer specific biological mark can comprise miR that one or more (for example 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Fig. 3.

Can assess one or more mammary cancer specific biological marks, thereby the mammary cancer specific biological marking is provided.For example, the described biological marking can comprise that one or more cross the miR of expression, includes but not limited to miR-21, miR-155, miR-206, miR-122a, miR-210, miR-21, miR-21, miR-155, miR-206, miR-122a, miR-210 or miR-21 or their any combination.

The described biological marking can also comprise that one or more express not enough miR, such as but not limited to let-7, miR-10b, miR-125a, miR-125b, miR-145, miR-143, miR-145, miR-16, let-7, let-7, let-7, miR-10b, miR-125a, miR-125b or miR-145 or their any combination.

The mRNA that can analyze can include but not limited to ER, PR, HER2, MUC1 or EGFR or their any combination.Sudden change (include but not limited to and KRAS, B-Raf or CYP2D6, or their the relevant sudden change of any combination) also can be as the mammary cancer specific biological mark from vesica.In addition, can include but not limited to hsp70, MART-1, TRP, HER2, hsp70, MART-1, TRP, HER2, ER, PR, III class b-tubulin or VEGFA as protein, part or the peptide of the biomarker from mammary cancer specificity vesica, or their any combination.In addition, can include but not limited to GAS5 as the snoRNA of the exosome biomarker of mammary cancer.Gene fusion thing ETV6-NTRK3 also can be as the biomarker of mammary cancer.

The present invention also provides the vesica separated, and it comprises one or more mammary cancer specific biological mark, for example ETV6-NTRK3; The perhaps listed biomarker for mammary cancer in Fig. 3 and Fig. 1.The composition of the vesica that comprises separation also is provided.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more mammary cancer specific biological mark, for example ETV6-NTRK3; The perhaps listed biomarker for mammary cancer in Fig. 3 and Fig. 1.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony is the mammary cancer specificity vesica of homogeneous basically or the biomarker of vesica comprise for example, in one or more mammary cancer specific biological marks (ETV6-NTRK3) or Fig. 3 and Fig. 1 listed to(for) mammary cancer.

For characterizing mammary cancer, can also detect for example, in one or more mammary cancer specific biological marks (ETV6-NTRK3) or Fig. 3 and Fig. 1 listed biomarker for mammary cancer by one or more systems as herein described.For example, detection system can comprise that one or more probes for example, with the listed biomarker for mammary cancer in one or more mammary cancer specific biological marks (ETV6-NTRK3) of one or more vesicas of detection of biological sample or Fig. 3 and Fig. 1.

Ovarian cancer

Ovarian cancer specific biological mark from vesica (for example can comprise one or more, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) miR that cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Fig. 4, and can be for setting up the ovarian cancer specific biological marking.For example, the described biological marking can comprise that one or more cross the miR of expression, such as, but not limited to miR-200a, miR-141, miR-200c, miR-200b, miR-21, miR-141, miR-200a, miR-200b, miR-200c, miR-203, miR-205, miR-214, miR-199 ^*or miR-215 or their any combination.In addition, the described biological marking can also comprise that one or more express not enough miR, such as but not limited to miR-199a, miR-140, miR-145, miR-100, miR-let-7 group or miR-125b-1 or their any combination.One or more mRNA that can be analyzed can include but not limited to ERCC1, ER, TOPO1, TOP2A, AR, PTEN, HER2/neu, CD24 or EGFR, or their any combination.

The ovarian cancer biomarker sudden change that can be assessed in vesica includes but not limited to any combination of KRAS sudden change, B-Raf sudden change or the specific sudden change of ovarian cancer.The protein that can be assessed in vesica, part or peptide can include but not limited to VEGFA, VEGFR2 or HER2, or their any combination.In addition, the vesica of separation or mensuration can be for ovarian cancer cell is specific or be derived from ovarian cancer cell.

The present invention also provides the vesica separated, and it comprises one or more ovarian cancer specific biological mark, for example CD24; The perhaps listed biomarker for ovarian cancer in Fig. 4 and Fig. 1.The composition of the vesica that comprises separation also is provided.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more ovarian cancer specific biological mark, for example CD24; The perhaps listed biomarker for ovarian cancer in Fig. 4 and Fig. 1.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony is the ovarian cancer specificity vesica of homogeneous basically or the vesica that comprises for example, in one or more ovarian cancer specific biological marks (CD24) or Fig. 4 and Fig. 1 listed those.

For characterizing ovarian cancer, can also detect for example, in one or more ovarian cancer specific biological marks (CD24) or Fig. 4 and Fig. 1 listed ovarian cancer specific biological mark by one or more systems as herein described.For example, detection system can comprise one or more probes, for example, with listed those in one or more ovarian cancer specific biological marks (CD24) of one or more vesicas in the detection of biological sample or Fig. 4 and Fig. 1.

Lung cancer

Lung cancer specific biological mark from vesica (for example can comprise one or more, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) miR that cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Fig. 5, and can be for setting up the lung cancer specific biological marking.

The described biological marking can comprise that one or more cross the miR of expression, such as but not limited to miR-21, miR-205, miR-221 (protectiveness), let-7a (protectiveness), miR-137 (risk), miR-372 (risk), miR-122a (risk) or their any combination.The described biological marking can comprise one or more rises or cross the miRNA expressed, for example miR-17-92, miR-19a, miR-21, miR-92, miR-155, miR-191, miR-205 or miR-210; One or more downwards or express not enough miRNA, for example miR-let-7, or their any combination.Described one or more biomarkers can be miR-92a-2 ^*, miR-147, miR-574-5p, as for small cell lung cancer.

One or more mRNA that can be analyzed can include but not limited to EGFR, PTEN, RRM1, RRM2, ABCB1, ABCG2, LRP, VEGFR2, VEGFR3, III class b-tubulin or their any combination.

The biomarker sudden change of the lung cancer that can be assessed in vesica includes but not limited to the sudden change of EGFR, KRAS, B-Raf, UGT1A1; Perhaps any combination of the specific sudden change of lung cancer.The protein that can be assessed in vesica, part or peptide can include but not limited to KRAS, hENT1 or their any combination.

Described biomarker can also be Midkine (MK or MDK).In addition, the vesica that separates or analyze can be for lung carcinoma cell is specific or be derived from lung carcinoma cell.

The present invention also provides the vesica separated, and it comprises one or more lung cancer specific biological mark, for example RLF-MYCL1, TGF-ALK or CD74-ROS1; Perhaps listed those in Fig. 5 and Fig. 1.The composition of the vesica that comprises separation also is provided.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more lung cancer specific biological mark, for example RLF-MYCL1, TGF-ALK or CD74-ROS1; Perhaps listed those in Fig. 5 and Fig. 1.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony is the lung cancer specificity vesica of homogeneous basically or the vesica that comprises for example, in one or more lung cancer specific biological marks (RLF-MYCL1, TGF-ALK or CD74-ROS 1) or Fig. 5 and Fig. 1 listed those.

For characterizing lung cancer, can also detect for example, in one or more lung cancer specific biological marks (RLF-MYCL1, TGF-ALK or CD74-ROS1) or Fig. 5 and Fig. 1 listed those biomarkers for lung cancer by one or more systems as herein described.For example, detection system can comprise that one or more probes for example, with listed those biomarkers for lung cancer in one or more lung cancer specific biological marks (RLF-MYCL1, TGF-ALK or CD74-ROS 1) of one or more vesicas in the detection of biological sample or Fig. 5 and Fig. 1.

Colorectal carcinoma

Colorectal carcinoma specific biological mark from vesica (for example can comprise one or more, 2, the miR 3,4,5,6,7,8 or more kinds of) cross expressed, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Fig. 6, and can be for setting up the colorectal carcinoma specific biological marking.For example, the described biological marking can comprise that one or more cross the miR of expression, such as but not limited to miR-24-1, miR-29b-2, miR-20a, miR-10a, miR-32, miR-203, miR-106a, miR-17-5p, miR-30c, miR-223, miR-126, miR-128b, miR-21, miR-24-2, miR-99b, miR-155, miR-213, miR-150, miR-107, miR-191, miR-221, miR-20a, miR-510, miR-92, miR-513, miR-19a, miR-21, miR-20, miR-183, miR-96, miR-135b, miR-31, miR-21, miR-92, miR-222, miR-181b, miR-210, miR-20a, miR-106a, miR-93, miR-335, miR-338, miR-133b, miR-346, miR-106b, miR-153a, miR-219, miR-34a, miR-99b, miR-185, miR-223, miR-211, miR-135a, miR-127, miR-203, miR-212, miR-95 or miR-17-5p or their any combination.The described biological marking can also comprise that one or more express not enough miR, miR-143 for example, miR-145, miR-143, miR-126, miR-34b, miR-34c, let-7, miR-9-3, miR-34a, miR-145, miR-455, miR-484, miR-101, miR-145, miR-133b, miR-129, miR-124a, miR-30-3p, miR-328, miR-106a, miR-17-5p, miR-342, miR-192, miR-1, miR-34b, miR-215, miR-192, miR-301, miR-324-5p, miR-30a-3p, miR-34c, miR-331, miR-548c-5p, miR-362-3p, miR-422a or miR-148b or their any combination.

Described one or more biomarkers can for raise or cross the miRNA (for example miR-20a, miR-21, miR-106a, miR-181b or miR-203) that expresses with for characterizing adenocarcinoma of colon.Described one or more biomarkers can, for characterizing colorectal carcinoma, for example raise or cross expression miRNA (described miRNA is selected from miR-19a, miR-21, miR-127, miR-31, miR-96, miR-135b and miR-183); Lower or express not enough miRNA (for example miR-30c, miR-133a, mirl43, miR-133b, miR-145) or their any combination.Described one or more biomarkers can be used for characterizing colorectal carcinoma, such as raising or cross the miRNA expressed, it is selected from: miR-548c-5p, miR-362-3p, miR-422a, miR-597, miR-429, miR-200a and miR-200b, or their any combination.

One or more mRNA that can be analyzed can include but not limited to EFNB1, ERCC1, HER2, VEGF or EGFR or their any combination.The biomarker sudden change of the colorectal carcinoma that can be assessed in vesica includes but not limited to the sudden change of EGFR, KRAS, VEGFA, B-Raf, APC or p53; Perhaps any combination of the specific sudden change of colorectal carcinoma.The protein that can be assessed in vesica, part or peptide can include but not limited to AFR, Rabs, ADAM10, CD44, NG2, ephrin-B1, MIF, b-catenin, adaptor protein, plakoglobin, half lactadherin-4, RACK1, four transmembrane proteins-8, FasL, TRAIL, A33, CEA, EGFR, pepx 1, hsc-70, four transmembrane proteins, ESCRT, TS, PTEN or TOPO1, or their any combination.In addition, the vesica that separates or analyze can be for colon cancer cell is specific or be derived from colon cancer cell.

The present invention also provides the vesica separated, and it comprises one or more colorectal carcinoma specific biological marks, for example the listed biomarker for colorectal carcinoma in Fig. 6 and Fig. 1.The composition of the vesica that comprises separation also is provided in addition.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more colorectal carcinoma specific biological marks, for example the listed biomarker for colorectal carcinoma in Fig. 6 and Fig. 1.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony is the colorectal carcinoma specificity vesica of homogeneous basically or the vesica that comprises one or more colorectal carcinoma specific biological marks (for example in Fig. 6 and Fig. 1 listed the biomarker for colorectal carcinoma).

For characterizing colorectal carcinoma, can also detect one or more colorectal carcinoma specific biological marks (for example in Fig. 6 and Fig. 1 listed the biomarker for colorectal carcinoma) by one or more systems as herein described.For example, detection system for example can comprise one or more probes, with one or more colorectal carcinoma specific biological marks of one or more vesicas in the detection of biological sample (in Fig. 6 and Fig. 1 listed the biomarker for colorectal carcinoma).

Adenoma is to hyperplastic polyp

Adenoma from vesica (for example can comprise one or more to hyperplastic polyp specific biological mark, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) miR that cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide or their any combination, for example listed in Fig. 7, and can be for setting up the specific biological marking of adenoma to hyperplastic polyp.For example, one or more mRNA that can analyze can include but not limited to ABCA8, KIAA1199, GCG, MAMDC2, C2orf32,229670_at, IGF1, PCDH7, PRDX6, PCNA, COX2 or MUC6, or their any combination.

Can in vesica, be assessed for distinguish adenoma to the biomarker of hyperplastic polyp sudden change include but not limited to KRAS sudden change, B-Raf sudden change or distinguish specifically any combination of the sudden change of adenoma and hyperplastic polyp.The protein that can be assessed in vesica, part or peptide can include but not limited to hTERT.

The present invention also provides the vesica separated, and it comprises one or more for distinguishing the specific biological mark of adenoma and hyperplastic polyp, for example listed in Fig. 7.The composition of the vesica that comprises separation also is provided in addition.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more for distinguishing the specific biological mark of adenoma and hyperplastic polyp, for example listed in Fig. 7.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony is the homogeneous basically with one or more specific biological marks for distinguishing adenoma and hyperplastic polyp, for example listed in Fig. 7.

For distinguishing adenoma and hyperplastic polyp, can also for example detect, for distinguishing one or more specific biological marks (Fig. 7 is listed) of adenoma and hyperplastic polyp by one or more systems as herein described.For example, detection system can comprise one or more probes with one or more vesicas in the detection of biological sample, for example, for distinguishing one or more specific biological marks (Fig. 7 is listed) of adenoma and hyperplastic polyp.

Bladder cancer

The biomarker of bladder cancer can be used for the method according to this invention assessment bladder cancer.Described biomarker can comprise miR that one or more (for example 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination.The biomarker of bladder cancer includes but not limited to one or more in miR-223, miR-26b, miR-221, miR-103-1, miR-185, miR-23b, miR-203, miR-17-5p, miR-23a, miR-205 or its arbitrary combination.Other biomarker of bladder cancer comprises FGFR3, EGFR, pRB (Retinoblastoma Protein), 5T4, p53, Ki-67, VEGF, CK20, COX2, p21, cyclin D1, p14, p15, p16, Her-2, MAPK (mitogen activated protein kinase), Bax/Bcl-2, PI3K (phosphoinositide-3-kinases), CDK (cell cycle protein dependent kinase), CD40, TSP-1, HA-ase, Telomerase, Survivin, NMP22, TNF, Cyclin E1, p27, caspase, Survivin, NMP22 (NMP-22), BCLA-4, cytokeratin (8, 18, 19 and 20), CYFRA 21-1, IL-2 and complement factor H associated protein.In one embodiment, the bladder cancer mark that acts on diagnosis, prognosis and treatment diagnostic purpose for nonreceptor tyrosine kinase ETK/BMX and/or carbonic anhydrase IX.Referring to, Guo etc., Tyrosine Kinase ETK/BMX Is Up-Regulated in Bladder Cancer and Predicts Poor Prognosis in Patients with Cystectomy.PLoS One.2011 March 7; 6 (3): e17778.; Klatte etc., Carbonic anhydrase IX in bladder cancer:a diagnostic, prognostic, and therapeutic molecular marker.Cancer.2009 April 1; 115 (7): 1448-58.Described biomarker can be one or more vesica biomarkers associated with bladder cancer, as is described in Pisitkun etc., Discovery of urinary biomarkers.Mol Cell Proteomics.2006 October; 5 (10): 1760-71; Welton etc., Proteomics analysis of bladder cancer exosomes.Mol Cell Proteomics.2010 June; 9 (6): in 1324-38.These biomarkers can be used for assessing bladder cancer.Described mark also can be associated with vesica or vesica colony.

Intestines easily swash disease (IBD)

IBD from vesica (for example can comprise one or more to normal biomarker, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) miR that cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Fig. 8, and can be for setting up IBD to the normal specific biological marking.One or more mRNA that for example, can analyze can include but not limited to REG1A, MMP3 or their any combination.

The present invention also provides the vesica separated, and it comprises one or more for distinguishing the specific biological mark of IBD and normal specimens, for example listed in Fig. 8.The composition of the vesica that comprises separation also is provided.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more for distinguishing the specific biological mark of IBD and normal specimens, for example listed in Fig. 8.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony is the homogeneous basically with one or more specific biological marks for distinguishing IBD and normal specimens, for example listed in Fig. 8.

For distinguishing IBD and normal specimens, can also for example detect, for distinguishing one or more specific biological marks (Fig. 8 is listed) of IBD and normal specimens by one or more systems as herein described.For example, detection system can comprise one or more probes with one or more vesicas in the detection of biological sample, for example, for distinguishing one or more specific biological marks (Fig. 8 is listed) of IBD and normal specimens.

Adenoma is to colorectal carcinoma (CRC)

Adenoma from vesica (for example can comprise one or more to the specific biomarker of CRC, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) miR that cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Fig. 9, and can be for setting up the specific biological marking of adenoma to CRC.One or more mRNA that for example, can analyze can include but not limited to GREM1, DDR2, GUCY1A3, TNS1, ADAMTS1, FBLN1, FLJ38028, RDX, FAM129A, ASPN, FRMD6, MCC, RBMS1, SNAI2, MEIS1, DOCK10, PLEKHC1, FAM126A, TBC1D9, VWF, DCN, ROBO1, MSRB3, LATS2, MEF2C, IGFBP3, GNB4, RCN3, AKAP12, RFTN1,226834_at, COL5A1, GNG2, NR3C1 ^*, SPARCL1, MAB21L2, AXIN2,236894_at, AEBP1, AP1S2, C10orf56, LPHN2, AKT3, FRMD6, COL15A1, CRYAB, COL14A1, LOC286167, QKI, WWTR1, GNG11, PAPPA or ELDT1 or their any combination.

The present invention also provides the vesica separated, and it comprises one or more for distinguishing the specific biological mark of adenoma and CRC, for example listed in Fig. 9.The composition of the vesica that comprises separation also is provided in addition.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more for distinguishing the specific biological mark of adenoma and CRC, for example listed in Fig. 9.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony is the homogeneous in fact with one or more specific biological marks for distinguishing adenoma and CRC, for example listed in Fig. 9.

For distinguishing adenoma and CRC, can also for example detect, for distinguishing one or more specific biological marks (Fig. 9 is listed) of adenoma and CRC by one or more systems as herein described.For example, detection system can comprise one or more probes with one or more vesicas in the detection of biological sample, for example, for distinguishing one or more specific biological marks (Fig. 9 is listed) of adenoma and CRC.

IBD is to CRC

IBD from vesica (for example can comprise one or more to CRC specific biological mark, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) miR that cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Figure 10, and can be for setting up the specific biological marking of IBD to CRC.One or more mRNA that for example, can analyze can include but not limited to 227458_at, INDO, CXCL9, CCR2, CD38, RARRES3, CXCL10, FAM26F, TNIP3, NOS2A, CCRL1, TLR8, IL18BP, FCRL5, SAMD9L, ECGF1, TNFSF13B, GBP5 or GBP1 or their any combination.

The present invention also provides the vesica separated, and it comprises one or more for distinguishing the specific biological mark of IBD and CRC, for example listed in Figure 10.The composition of the vesica that comprises separation also is provided in addition.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more for distinguishing the specific biological mark of IBD and CRC, for example listed in Figure 10.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony is the homogeneous in fact with one or more specific biological marks for distinguishing IBD and CRC, for example listed in Figure 10.

For distinguishing IBD and CRC, can also for example detect, for distinguishing one or more specific biological marks (Figure 10 is listed) of IBD and CRC by one or more systems as herein described.For example, detection system can comprise one or more probes with one or more vesicas in the detection of biological sample, for example, for distinguishing one or more specific biological marks (Figure 10 is listed) of IBD and CRC.

CRC Dukes B is to Dukes C-D

CRC Dukes B from vesica (for example can comprise one or more to Dukes C-D specific biological mark, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) miR that cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Figure 11, and can be for setting up the specific biological marking of CRC Dukes B to C-D.One or more mRNA that for example, can analyze can include but not limited to TMEM37 ^*, IL33, CA4, CCDC58, CLIC6, VERSUSNL1, ESPN, APCDD1, C13orf18, CYP4X1, ATP2A3, LOC646627, MUPCDH, ANPEP, C1orf115, HSD3B2, GBA3, GABRB2, GYLTL1B, LYZ, SPC25, CDKN2B, FAM89A, MOGAT2, SEMA6D, 229376_at, TSPAN5, IL6R or SLC26A2 or their any combination.

The present invention also provides the vesica separated, and it comprises one or more for distinguishing the specific biological mark of CRC Dukes B and CRC Dukes C-D, for example listed in Figure 11.The composition of the vesica that comprises separation also is provided in addition.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more for distinguishing the specific biological mark of CRC Dukes B and CRCDukes C-D, for example listed in Figure 11.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony is the homogeneous basically with one or more specific biological marks for distinguishing CRC Dukes B and CRC Dukes C-D, for example listed in Figure 11.

For distinguishing CRC Dukes B and CRC Dukes C-D, can also for example detect, for distinguishing one or more specific biological marks (Figure 11 is listed) of CRC Dukes B and CRC Dukes C-D by one or more systems as herein described.For example, detection system can comprise one or more probes with one or more vesicas in the detection of biological sample, for example, for distinguishing one or more specific biological marks (Figure 11 is listed) of CRC Dukes B and CRC Dukes C-D.

There is the adenoma of low dysplasia to thering is the adenoma of high grade dysplasia

(for example can comprise one or more from the adenoma with low dysplasia of vesica to the specific biological mark of adenoma with high grade dysplasia, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) miR that cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Figure 12, and the adenoma that can have a low dysplasia for foundation is to the specific biological marking of adenoma with high grade dysplasia.One or more mRNA that for example, can analyze can include but not limited to SI, DMBT1, CFI ^*, AQP1, APOD, TNFRSF17, CXCL10, CTSE, IGHA1, SLC9A3, SLC7A1, BATF2, SOCS1, DOCK2, NOS2A, HK2, CXCL2, IL15RA, POU2AF1, CLEC3B, ANI3BP, MGC13057, LCK ^*, C4BPA, HOXC6, GOLT1A, C2orf32, IL10RA, 240856_at, SOCS3, MEIS3P1, HIPK1, GLS, CPLX1, 236045_x_at, GALC, AMN, CCDC69, CCL28, CPA3, TRIB2, HMGA2, PLCL2, NR3C 1, EIF5A, LARP4, RP5-1022P6.2, PHLDB2, FKBP1B, INDO, CLDN8, CNTN3, PBEF1, SLC16A9, CDC25B, TPSB2, PBEF1, ID4, GJB5, CHN2, LIMCH1 or CXCL9 or their any combination.

The present invention also provides the vesica separated, and it comprises one or more and has the adenoma of low dysplasia and the specific biological mark with adenoma of high grade dysplasia for differentiation, for example listed in Figure 12.The composition of the vesica that comprises separation also is provided in addition.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more and has the adenoma of low dysplasia and the specific biological mark with adenoma of high grade dysplasia for differentiation, for example listed in Figure 12.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony is (for example Figure 12 the is listed) homogeneous basically with the adenoma that has a low dysplasia for differentiation and one or more specific biological marks of the adenoma with high grade dysplasia.

For differentiation has the adenoma of low dysplasia and has the adenoma of high grade dysplasia, can also detect for differentiation and there are the adenoma of low dysplasia and one or more specific biological marks (for example Figure 12 is listed) with adenoma of high grade dysplasia by one or more systems as herein described.For example, detection system can comprise one or more probes with one or more vesicas in the detection of biological sample, there are the adenoma of low dysplasia and one or more specific biological marks (for example Figure 12 is listed) with adenoma of high grade dysplasia for differentiation.

Ulcerative colitis (UC) is to Crohn disease (CD)

(for example can comprise one or more from the ulcerative colitis (UC) of vesica to the specific biological mark of Crohn disease (CD), 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) miR that cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Figure 13, and can be for setting up the specific biological marking of UC to CD.One or more mRNA that for example, can analyze can include but not limited to IFITM1, IFITM3, STAT1, STAT3, TAP1, PSME2, PSMB8, HNF4G, KLF5, AQP8, APT2B1, SLC16A, MFAP4, CCNG2, SLC44A4, DDAH1, TOB1,231152_at, MKNK1, CEACAM7 ^*, 1562836_at, CDC42SE2, PSD3,231169_at, IGL@ ^*, GSN, GPM6B, CDV3 ^*, PDPK1, ANP32E, ADAM9, CDH1, NLRP2,215777_at, OSBPL1, VNN1, RABGAP1L, PHACTR2, ASH1L, 213710_s_at, CDH1, NLRP2,215777_at, OSBPL1, VNN1, RABGAP1L, PHACTR2, ASH1,213710_s_at, ZNF3, FUT2, IGHA1, EDEM1, GPR171,229713_at, LOC643187, FLVCR1, SNAP23 ^*, ETNK1, LOC728411, POSTN, MUC12, HOXA5, SIGLEC1, LARP5, PIGR, SPTBN1, UFM1, C6orf62, WDR90, ALDH1A3, F2RL1, IGHV1-69, DUOX2, RAB5A or CP; Perhaps their any combination also can be as the specific biological mark to CD from the UC of vesica.

The biomarker sudden change for distinguishing UC and CD that can be assessed in vesica includes but not limited to the sudden change of CARD15 or distinguishes specifically any combination of the sudden change of UC and CD.The protein that can be assessed in vesica, part or peptide can include but not limited to (P) ASCA.

The present invention also provides the vesica separated, and it comprises one or more for distinguishing the specific biological mark of UC and CD, for example listed in Figure 13.The composition of the vesica that comprises separation also is provided in addition.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more for distinguishing the specific biological mark of UC and CD, for example listed in Figure 13.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony for example, is homogeneous basically for having one or more specific biological marks (Figure 13 is listed) for distinguishing UC and CD.

For distinguishing UC and CD, can also for example detect, for distinguishing one or more specific biological marks (Figure 13 is listed) of UC and CD by one or more systems as herein described.For example, detection system can comprise one or more probes with one or more vesicas in the detection of biological sample, for example, for distinguishing one or more specific biological marks (Figure 13 is listed) of UC and CD.

Hyperplastic polyp

Hyperplastic polyp from vesica (for example can comprise one or more to normal specific biological mark, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) miR that cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Figure 14, and can be for setting up hyperplastic polyp to the normal specific biological marking.One or more mRNA that for example, can analyze can include but not limited to SLC6A14, ARHGEF10, ALS2, IL1RN, SPRY4, PTGER3, TRIM29, SERPINB5,1560327_at, ZAK, BAG4, TRIB3, TTL, FOXQ1 or any combination.

The present invention also provides the vesica separated, and the specific biological mark that it comprises one or more hyperplastic polyps is for example listed in Figure 14.The composition of the vesica that comprises separation also is provided.Therefore, in some embodiments, described composition comprises vesica colony, and the specific biological mark that this colony comprises one or more hyperplastic polyps is for example listed in Figure 14.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony for example, is homogeneous basically for hyperplastic polyp specificity vesica or the vesica that comprises one or more hyperplastic polyp specific biological marks (listed in Figure 14).

For characterizing hyperplastic polyp, can also detect by one or more systems as herein described the specific biological mark (for example listed in Figure 14) of one or more hyperplastic polyps.For example, detection system can comprise that one or more probes are to detect listed one or more in Figure 14.One or more hyperplasia specific biological marks (for example listed in Figure 14) of one or more vesicas in biological sample.

Adenoma with low dysplasia is to normally

The adenoma with low dysplasia from vesica (for example can comprise one or more to normal specific biological mark, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) miR that cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Figure 15, and can there is the adenoma of low dysplasia to the normal specific biological marking for foundation.For example, the RNA that can analyze can include but not limited to UGT2A3, KLK11, KIAA1199, FOXQ1, CLDN8, ABCA8 or PYY or their any combination, and can be used as from the low dysplasia of having of vesica normal specific biological mark.In addition, can include but not limited to GAS5 as thering is the snoRNA of low dysplasia to normal exosome biomarker.

The present invention also provides the vesica separated, and it comprises one or more adenomas that have low dysplasia for differentiation and normal specific biological mark, for example listed in Figure 15.The composition of the vesica that comprises separation also is provided.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more adenomas that have low dysplasia for differentiation and normal specific biological mark, for example listed in Figure 15.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony for example, for to have the adenoma that has a low dysplasia for differentiation and normal one or more specific biological marks (Figure 15 is listed) be homogeneous basically.

In addition, the adenoma that has a low dysplasia for differentiation, with normal, can also detect the adenoma and normal one or more specific biological marks (for example Figure 15 is listed) that has low dysplasia for differentiation by one or more systems as herein described.For example, detection system can comprise that one or more probes for example, with one or more vesicas in the detection of biological sample, adenoma have low dysplasia for differentiation and normal one or more specific biological marks (Figure 15 is listed).

Adenoma is to normally

Adenoma from vesica (for example can comprise one or more to normal specific biological mark, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) miR that cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Figure 16, and can be for setting up adenoma to the normal specific biological marking.One or more RNA that for example, can analyze can include but not limited to KIAA1199, FOXQ1 or CA7 or their any combination.Can include but not limited to clusterin as protein, part or the peptide from the biomarker of vesica (its to adenoma to normally thering is specificity).

The present invention also provides the vesica separated, and it comprises one or more for distinguishing adenoma and normal specific biological mark, for example listed in Figure 16.The composition of the vesica that comprises separation also is provided in addition.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more for distinguishing adenoma and normal specific biological mark, for example listed in Figure 16.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony is for for example having, for to distinguish adenoma and normal one or more specific biological marks (Figure 16 is listed) be homogeneous basically.

For distinguishing adenoma with normal, can also for example detect, for distinguishing adenoma and normal one or more specific biological marks (Figure 16 is listed) by one or more systems as herein described.For example, detection system can comprise one or more probes with one or more vesicas in the detection of biological sample, for example, for distinguishing adenoma and normal one or more specific biological marks (Figure 16 is listed).

CRC is to normally

CRC from vesica (for example can comprise one or more to normal specific biological mark, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) miR that cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Figure 17, and can be for setting up CRC to the normal specific biological marking.For example, one or more mRNA that can analyze can include but not limited to VWF, IL8, CHI3L1, S100A8, GREM1 or ODC or their any combination, and can be as the CRC from vesica to normal specific biological mark.

That can in vesica, be assessed suddenlys change and includes but not limited to the sudden change of KRAS, BRAF, APC, MSH2 or MLH1 normal biomarker for CRC; Perhaps distinguish specifically CRC and the normal any combination suddenlyd change.The protein that can be assessed in vesica, part or peptide can include but not limited to CK13, calcineurin (clacineurin), CHK1, clathrin light-chain, phosphoric acid-ERK, phosphoric acid-PTK2 or MDM2 or their any combination.

The present invention also provides the vesica separated, and it comprises one or more for distinguishing CRC and normal specific biological mark, for example listed in Figure 17.The composition of the vesica that comprises separation also is provided in addition.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more for distinguishing CRC and normal specific biological mark, for example listed in Figure 17.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony is for for example having, for to distinguish CRC and normal one or more specific biological marks (Figure 17 is listed) be homogeneous basically.

For distinguishing CRC with normal, can also for example detect, for distinguishing CRC and normal one or more specific biological marks (Figure 17 is listed) by one or more systems as herein described.For example, detection system can comprise one or more probes with one or more vesicas in the detection of biological sample, for example, for distinguishing CRC and normal one or more specific biological marks (Figure 17 is listed).

Benign prostatic hyperplasia (BPH)

Benign prostatic hyperplasia (BPH) specific biological mark from vesica (for example can comprise one or more, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) miR that cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Figure 18, and can be for setting up the BPH specific biological marking.The protein that can be assessed in vesica, part or peptide can include but not limited to complete fibronectin.

The present invention also provides the vesica separated, and it comprises one or more BPH specific biological marks, for example in Figure 18 and Fig. 1 for BPH listed biomarker.The composition of the vesica that comprises separation also is provided.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more BPH specific biological marks, for example the listed biomarker for BPH in Figure 18 and Fig. 1.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony is for example, to be homogeneous basically for BPH specificity vesica or the vesica that comprises one or more BPH specific biological marks (in Figure 18 and Fig. 1 listed the biomarker for BPH).

For characterizing BPH, can also detect one or more BPH specific biological marks (for example in Figure 18 and Fig. 1 listed those biomarkers for BPH) by one or more systems as herein described.For example, detection system can comprise that one or more probes for example, with one or more BPH specific biological marks of one or more vesicas in the detection of biological sample (in Figure 18 and Fig. 1 listed those biomarkers for BPH).

Prostate cancer

Prostatic cancer specific biomarker from vesica (for example can comprise one or more, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) miR that cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Figure 19, and can be for setting up the biological marking of prostatic cancer specific.For example, the biological marking of prostate cancer can comprise miR-9, miR-21, miR-141, miR-370, miR-200b, miR-210, miR-155 or miR-196a.In some embodiments, the described biological marking can comprise that one or more cross the miR of expression, such as but not limited to miR-202, miR-210, miR-296, miR-320, miR-370, miR-373, miR-498, miR-503, miR-184, miR-198, miR-302c, miR-345, miR-491, miR-513, miR-32, miR-182, miR-31, miR-26a-1/2, miR-200c, miR-375, miR-196a-1/2, miR-370, miR-425, miR-425, miR-194-1/2, miR-181a-1/2, miR-34b, let-7i, miR-188, miR-25, miR-106b, miR-449, miR-99b, miR-93, miR-92-1/2, miR-125a or miR-141 or their any combination.

The described biological marking can also comprise that one or more express not enough miR, such as but not limited to let-7a, let-7b, let-7c, let-7d, let-7g, miR-16, miR-23a, miR-23b, miR-26a, miR-92, miR-99a, miR-103, miR-125a, miR-125b, miR-143, miR-145, miR-195, miR-199, miR-221, miR-222, miR-497, let-7f, miR-19b, miR-22, miR-26b, miR-27a, miR-27b, miR-29a, miR-29b, miR-30_5p, miR-30c, miR-100, miR-141, miR-148a, miR-205, miR-520h, miR-494, miR-490, miR-133a-1, miR-1-2, miR-218-2, miR-220, miR-128a, miR-221, miR-499, miR-329, miR-340, miR-345, miR-410, miR-126, miR-205, miR-7-1/2, miR-145, miR-34a, miR-487 or et-7b or their any combination.The described biological marking can comprise the miR-21 that raises or cross expression; Lower or express not enough miR-15a, miR-16-1, miR-143 or miR-145; Or their any combination.

One or more mRNA that can analyze can include but not limited to AR, PCA3 or their any combination, and can be as the specific biological mark of the prostate cancer from vesica.

The protein that can be assessed in vesica, part or peptide can include but not limited to FASLG, TNFSF10 or their any combination.In addition, separate or the vesica analyzed can be for prostate gland cancer cell specificity, or be derived from prostate cancer cell.In addition, can include but not limited to U50 as the snoRNA of the exosome biomarker of prostate cancer.The example of the biological marking of prostate cancer further describes hereinafter.

The present invention also provides the vesica separated, it comprises one or more prostatic cancer specific biomarkers, for example ACSL3-ETV1, C15ORF21-ETV1, FLJ35294-ETV1, HERV-ETV1, TMPRSS2-ERG, TMPRSS2-ETV1/4/5, TMPRSS2-ETV4/5, SLC5A3-ERG, SLC5A3-ETV1, SLC5A3-ETV5 or KLK2-ETV4; The perhaps listed biomarker for prostate cancer in Figure 19, Figure 60 and Fig. 1.In some embodiments, the vesica of described separation is EpCam+, CK+, CD45-.The composition of the vesica that comprises separation also is provided.Therefore, in some embodiments, described composition comprises vesica colony, this colony comprises one or more prostatic cancer specific biomarkers, for example ACSL3-ETV1, C15ORF21-ETV1, FLJ35294-ETV1, HERV-ETV1, TMPRSS2-ERG, TMPRSS2-ETV1/4/5, TMPRSS2-ETV4/5, SLC5A3-ERG, SLC5A3-ETV1, SLC5A3-ETV5 or KLK2-ETV4; Perhaps listed those biomarkers for prostate cancer in Figure 19, Figure 60 and Fig. 1.In some embodiments, described composition comprises vesica colony, and it is EpCam+, CK+, CD45-.Described composition can comprise the vesica colony of obvious enrichment, wherein said vesica colony is for the prostatic cancer specific vesica or comprise for example ACSL3-ETV1 of one or more prostatic cancer specific biomarkers, C15ORF21-ETV1, FLJ35294-ETV1, HERV-ETV1, TMPRSS2-ERG, TMPRSS2-ETV1/4/5, TMPRSS2-ETV4/5, SLC5A3-ERG, SLC5A3-ETV1, SLC5A3-ETV5 or KLK2-ETV4 or Figure 19, in Figure 60 and Fig. 1, the vesica of listed those biomarkers for prostate cancer is homogeneous basically.In one embodiment, the vesica colony that described composition comprises obvious enrichment, it is EpCam+, CK+, CD45-.

For characterizing prostate cancer, can also detect one or more prostatic cancer specific biomarkers (for example ACSL3-ETV1, C15ORF21-ETV1, FLJ35294-ETV1, HERV-ETV1, TMPRSS2-ERG, TMPRSS2-ETV1/4/5, TMPRSS2-ETV4/5, SLC5A3-ERG, SLC5A3-ETV1, SLC5A3-ETV5 or KLK2-ETV4 by one or more systems as herein described; Perhaps listed those biomarkers for prostate cancer in Figure 19, Figure 60 and Fig. 1).In some embodiments, for characterizing prostate cancer, by one or more system detection of biological marks EpCam disclosed herein, CK (cytokeratin) and CD45, such as determining of the prostate cancer for the experimenter or experimenter's treatment resistance.For example, detection system can comprise that one or more probes for example, with one or more prostatic cancer specific biomarkers of one or more vesicas in the detection of biological sample (ACSL3-ETV1, C15ORF21-ETV1, FLJ35294-ETV1, HERV-ETV1, TMPRSS2-ERG, TMPRSS2-ETV1/4/5, TMPRSS2-ETV4/5, SLC5A3-ERG, SLC5A3-ETV1, SLC5A3-ETV5 or KLK2-ETV4; Perhaps listed those biomarkers for prostate cancer in Figure 19, Figure 60 and Fig. 1).In one embodiment, described detection system can comprise one or more probes for detection of EpCam, CK, CD45 or its combination.

Melanoma

Melanoma specific biological mark from vesica (for example can comprise one or more, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) miR that cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Figure 20, and can be for setting up the melanoma specific biological marking.For example, the described biological marking can comprise that one or more cross the miR of expression, such as but not limited to miR-19a, miR-144, miR-200c, miR-211, miR-324-5p, miR-331 or miR-374 or their any combination.The described biological marking can also comprise expresses not enough miR, such as but not limited to miR-9, miR-15a, miR-17-3p, miR-23b, miR-27a, miR-28, miR-29b, miR-30b, miR-31, miR-34b, miR-34c, miR-95, miR-96, miR-100, miR-104, miR-105, miR-106a, miR-107, miR-122a, miR-124a, miR-125b, miR-127, miR-128a, miR-128b, miR-129, miR-135a, miR-135b, miR-137, miR-138, miR-139, miR-140, miR-141, miR-149, miR-154, miR-154#3, miR-181a, miR-182, miR-183, miR-184, miR-185, miR-189, miR-190, miR-199, miR-199b, miR-200a, miR-200b, miR-204, miR-213, miR-215, miR-216, miR-219, miR-222, miR-224, miR-299, miR-302a, miR-302b, miR-302c, miR-302d, miR-323, miR-325, let-7a, let-7b, let-7d, let-7e or let-7g or their any combination.

One or more mRNA that can analyze can include but not limited to MUM-1, beta-catenin or Nop/5/Sik or their any combination, and can be as the melanoma specific biological mark from vesica.

The melanomatous biomarker sudden change that can be assessed in vesica includes but not limited to the sudden change of CDK4 or any combination of melanoma specific mutant.The protein that can be assessed in vesica, part or peptide can include but not limited to DUSP-1, Alix, hsp70, Gib2, Gia, moesin, GAPDH, malate dehydrogenase (malic acid dehydrogenase), p120 catenin, PGRL, syntaxin Jie Hedanbai1 & 2, born of the same parents are split albumen-2 or are contained WD repetitive proteins 1 or their any combination.Can include but not limited to H/ACA (U107f), SNORA11D or their any combination as the snoRNA of melanomatous exosome biomarker.In addition, what the vesica that separates or analyze can be for melanoma cell specific, or be derived from melanoma cell.

The present invention also provides the vesica separated, and it comprises one or more melanoma specific biological marks, for example listed for melanomatous biomarker in Figure 20 and Fig. 1.The composition of the vesica that comprises separation also is provided.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more melanoma specific biological marks, for example listed for melanomatous biomarker in Figure 20 and Fig. 1.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony for example, is homogeneous basically for melanoma specificity vesica or the vesica that comprises one or more melanoma specific biological marks (in Figure 20 and Fig. 1 listed for melanomatous biomarker).

In order to characterize melanoma, can also detect one or more melanoma specific biological marks (for example in Figure 20 and Fig. 1 listed for melanomatous biomarker) by one or more systems as herein described.For example, detection system can comprise that one or more probes for example, with one or more cancer specific biomarkers of one or more vesicas in the detection of biological sample (in Figure 20 and Fig. 1 listed for melanomatous biomarker).

Carcinoma of the pancreas

Carcinoma of the pancreas specific biological mark from vesica (for example can comprise one or more, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) miR that cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Figure 21, and can be for setting up the carcinoma of the pancreas specific biological marking.For example, the described biological marking can comprise that one or more cross the miR of expression, such as but not limited to miR-221, miR-181a, miR-155, miR-210, miR-213, miR-181b, miR-222, miR-181b-2, miR-21, miR-181b-1, miR-220, miR-181d, miR-223, miR-100-1/2, miR-125a, miR-143, miR-10a, miR-146, miR-99, miR-100, miR-199a-1, miR-10b, miR-199a-2, miR-221, miR-181a, miR-155, miR-210, miR-213, miR-181b, miR-222, miR-181b-2, miR-21, miR-181b-1, miR-181c, miR-220, miR-181d, miR-223, miR-100-1/2, miR-125a, miR-143, miR-10a, miR-146, miR-99, miR-100, miR-199a-1, miR-10b, miR-199a-2, miR-107, miR-103, miR-103-2, miR-125b-1, miR-205, miR-23a, miR-221, miR-424, miR-301, miR-100, miR-376a, miR-125b-1, miR-21, miR-16-1, miR-181a, miR-181c, miR-92, miR-15, miR-155, let-7f-1, miR-212, miR-107, miR-024-1/2, miR-18a, miR-31, miR-93, miR-224 or let-7d or their any combination.

The described biological marking can also comprise that one or more express not enough miR, such as but not limited to miR-148a, miR-148b, miR-375, miR-345, miR-142, miR-133a, miR-216, miR-217 or miR-139 or their any combination.One or more mRNA that can analyze can include but not limited to PSCA, mesothelin or osteopontin or their any combination, and can be as the carcinoma of the pancreas specific biological mark from vesica.

The biomarker sudden change of the carcinoma of the pancreas that can be assessed in vesica includes but not limited to the sudden change of KRAS, CTNNLB1, AKT, NCOA3 or B-RAF; Perhaps any combination of carcinoma of the pancreas specific mutant.Described biomarker can also be BRCA2, PALB2 or p16.In addition, separation or the vesica of analyzing can be specific for pancreatic cancer cell, or are derived from pancreatic cancer cell.

The present invention also provides the vesica separated, and it comprises one or more carcinoma of the pancreas specific biological marks, for example listed in Figure 21.The composition of the vesica that comprises separation also is provided.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more carcinoma of the pancreas specific biological marks, for example listed in Figure 21.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony for example, is homogeneous basically for carcinoma of the pancreas specificity vesica or the vesica that comprises one or more carcinoma of the pancreas specific biological marks (listed in Figure 21).

For characterizing carcinoma of the pancreas, can also detect one or more carcinoma of the pancreas specific biological marks (for example listed in Figure 21) by one or more systems as herein described.For example, detection system can comprise one or more probes one or more carcinoma of the pancreas specific biological marks (for example listed in Figure 21) with one or more vesicas in the detection of biological sample.

The cancer of the brain

The cancer of the brain (including but not limited to neurospongioma, glioblastoma, meningioma, acoustic tumor/schwannoma, medulloblastoma) specific biological mark from vesica (for example can comprise one or more, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) miR that cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Figure 22, and can be for setting up the cancer of the brain specific biological marking.For example, the described biological marking can comprise that one or more cross the miR of expression, such as but not limited to miR-21, miR-10b, miR-130a, miR-221, miR-125b-1, miR-125b-2, miR-9-2, miR-21, miR-25 or miR-123 or their any combination.

The described biological marking can also comprise that one or more express not enough miR, such as but not limited to miR-128a, miR-181c, miR-181a or miR-181b or their any combination.One or more mRNA that can analyze can include but not limited to MGMT, and it can be as the mark of the specific biological for the cancer of the brain from vesica.The protein that can be assessed in vesica, part or peptide can include but not limited to EGFR.

The present invention also provides the vesica separated, and it comprises one or more cancer of the brain specific biological mark, for example GOPC-ROS1; The perhaps listed biomarker for the cancer of the brain in Figure 22 and Fig. 1.The composition of the vesica that comprises separation also is provided.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more cancer of the brain specific biological mark, for example GOPC-ROS1; The perhaps listed biomarker for the cancer of the brain in Figure 22 and Fig. 1.Described composition can comprise the vesica colony of obvious enrichment, wherein said vesica colony's cancer of the brain specificity vesica or comprise one or more cancer of the brain specific biological marks for example the vesica of the listed biomarker for the cancer of the brain is homogeneous basically in GOPC-ROS1 or Figure 22 and Fig. 1.

For characterizing the cancer of the brain, can also detect one or more cancer of the brain specific biological marks (for example listed for the cancer of the brain in Figure 22 and Fig. 1) by one or more systems as herein described.For example, detection system can comprise that one or more probes for example, with listed those biomarkers for the cancer of the brain in one or more cancer of the brain specific biological marks (GOPC-ROS1) of one or more vesicas in the detection of biological sample or Figure 22 and Fig. 1.

Psoriatic

Psoriatic specific biological mark from vesica (for example can comprise one or more, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) miR that cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Figure 23, and can be for setting up the psoriatic specific biological marking.For example, the described biological marking can comprise the miR that one or more cross expression, such as but not limited to miR-146b, miR-20a, miR-146a, miR-31, miR-200a, miR-17-5p, miR-30e-5p, miR-141, miR-203, miR-142-3p, miR-21 or miR-106a or their any combination.The described biological marking can also comprise one or more and express not enough miR, such as but not limited to miR-125b, miR-99b, miR-122a, miR-197, miR-100, miR-381, miR-518b, miR-524, let-7e, miR-30c, miR-365, miR-133b, miR-10a, miR-133a, miR-22, miR-326 or miR-215 or their any combination.

One or more mRNA that can analyze can include but not limited to IL-20, VEGFR-1, VEGFR-2, VEGFR-3 or EGR1 or their any combination, and can be as the psoriatic specific biological mark from vesica.The psoriatic biomarker sudden change that can be assessed in vesica includes but not limited to the sudden change of MGST2; Perhaps any combination of the specific sudden change of psoriatic.

The present invention also provides the vesica separated, and it comprises one or more psoriatic specific biological marks, for example listed for psoriatic biomarker in Figure 23 and Fig. 1.The composition of the vesica that comprises separation also is provided.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more psoriatic specific biological marks, for example listed for psoriatic in Figure 23 and Fig. 1.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony for example, is homogeneous basically for psoriatic specificity vesica or the vesica that comprises one or more psoriatic specific biological marks (listed for psoriatic in Figure 23 and Fig. 1).

For for characterizing psoriatic, can also detect one or more psoriatic specific biological marks (for example listed for psoriatic in Figure 23 and Fig. 1) by one or more systems as herein described.For example, detection system can comprise one or more probes one or more psoriatic specific biological marks (for example listed for psoriatic in Figure 23 and Fig. 1) with one or more vesicas in the detection of biological sample.

Cardiovascular disorder (CVD)

CVD specific biological mark from vesica (for example can comprise one or more, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) miR that cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Figure 24, and can be for setting up the CVD specific biological marking.For example, the described biological marking can comprise that one or more cross the miR of expression, such as but not limited to miR-195, miR-208, miR-214, let-7b, let-7c, let-7e, miR-15b, miR-23a, miR-24, miR-27a, miR-27b, miR-93, miR-99b, miR-100, miR-103, miR-125b, miR-140, miR-145, miR-181a, miR-191, miR-195, miR-199a, miR-320, miR-342, miR-451 or miR-499 or their any combination.

The described biological marking can also comprise that one or more express not enough miR, such as but not limited to miR-1, miR-10a, miR-17-5p, miR-19a, miR-19b, miR-20a, miR-20b, miR-26b, miR-28, miR-30e-5p, miR-101, miR-106a, miR-126, miR-222, miR-374, miR-422b or miR-423 or their any combination.The mRNA that can analyze can include but not limited to MRP14, CD69 or their any combination, and can be as the specific biological mark of the CVD from vesica.

The biomarker sudden change of the CVD that can be assessed in vesica includes but not limited to the sudden change of MYH7, SCN5A or CHRM2; Perhaps any combination of the specific mutant of CVD.

The protein that can be assessed in vesica, part or peptide can include but not limited to CK-MB, cTnI (cardiac troponin), CRP, BPN, IL-6, MCSF, CD40, CD40L or their any combination.In addition, separate or the vesica analyzed can be for the CVD cell-specific, or be derived from the myocardial cell's.

The present invention also provides the vesica separated, and it comprises one or more CVD specific biological marks, for example listed for CVD in Figure 24 and Fig. 1.The composition of the vesica that comprises separation also is provided.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more CVD specific biological marks, for example listed for CVD in Figure 24 and Fig. 1.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony for example, is homogeneous basically for CVD specificity vesica or the vesica that comprises one or more CVD specific biological marks (in Figure 24 and Fig. 1 for CVD listed biomarker).

For characterizing CVD, can also detect one or more CVD specific biological marks (for example listed for CVD in Figure 24 and Fig. 1) by one or more systems disclosed herein.For example, detection system can comprise one or more probes one or more CVD specific biological marks (for example listed for CVD in Figure 24 and Fig. 1) with one or more vesicas in the detection of biological sample.

The increase (as disclosed in US publication No.2010/0010073) of miRNA or miRNA combination (such as miR-21, miR-129, miR-212, miR-214, miR-134 or its combination) can be used for diagnosis cardiac hypertrophy and/or risk increase or cardiac hypertrophy and/or existence already in heart failure in heart failure occurs.The downward of miR-182, miR-290 or its combination can be used for diagnosis cardiac hypertrophy and/or risk increase or cardiac hypertrophy and/or existence already in heart failure in heart failure occurs.The expression increase of miR-21, miR-129, miR-212, miR-214, miR-134 or its combination and the expression of miR-182, miR-290 or its combination reduce risk increase or cardiac hypertrophy and/or the existence in heart failure that can be used for diagnosis generation cardiac hypertrophy and/or heart failure.

Leukemia

Hematologic malignancies specific biological mark from vesica (for example can comprise one or more, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) miR that cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Figure 25, and can be for setting up the hematologic malignancies specific biological marking.For example, one or more mRNA that can analyze can include but not limited to HOX11, TAL1, LY1, LMO1 or LMO2 or their any combination, and can be as the specific biological mark of the hematologic malignancies from vesica.

The biomarker sudden change of the leukemia that can be assessed in vesica includes but not limited to c-kit, PDGFR or ABL sudden change; Perhaps any combination of hematologic malignancies specific mutant.

The present invention also provides the vesica separated, and it comprises one or more leukemia specific biological marks, for example listed for leukemia in Figure 25 and Fig. 1.The composition of the vesica that comprises separation also is provided.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more leukemia specific biological marks, for example listed for leukemia in Figure 25 and Fig. 1.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony for example, is homogeneous basically for leukemia specificity vesica or the vesica that comprises one or more leukemia specific biological marks (listed for leukemia in Figure 25 and Fig. 1).

For characterizing leukemia, can also detect one or more leukemia specific biological marks (for example listed for leukemia in Figure 25 and Fig. 1) by one or more systems disclosed herein.For example, detection system can comprise one or more probes one or more leukemia specific biological marks (for example listed for leukemia in Figure 25 and Fig. 1) with one or more vesicas in the detection of biological sample.

Described one or more leukemia specific biological marks can also be genetic fusant, and it is selected from TTL-ETV6, CDK6-MLL, CDK6-TLX3, ETV6-FLT3, ETV6-RUNX1, ETV6-TTL, MLL-AFF1, MLL-AFF3, MLL-AFF4, MLL-GAS7, TCBA1-ETV6, TCF3-PBX1 or TCF3-TFPT for kemia (ALL), be selected from BCL11B-TLX3, IL2-TNFRFS17, NUP214-ABL1, NUP98-CCDC28A, TAL1-STIL or ETV6-ABL2 for T cell acute lymphoblastic leukemia (T-ALL), be selected from ATIC-ALK, KIAA1618-ALK, MSN-ALK, MYH9-ALK, NPM1-ALK, TGF-ALK or TPM3-ALK for primary cutaneous type (ALCL), be selected from BCR-ABL1, BCR-JAK2, ETV6-EVI1, ETV6-MN1 or ETV6-TCBA1 for chronic lymphocytic leukemia (CML), be selected from CBFB-MYH11 for AML, CHIC2-ETV6, ETV6-ABL1, ETV6-ABL2, ETV6-ARNT, ETV6-CDX2, ETV6-HLXB9, ETV6-PER1, MEF2D-DAZAP1, AML-AFF1, MLL-ARHGAP26, MLL-ARHGEF12, MLL-CASC5, MLL-CBL, MLL-CREBBP, MLL-DAB21P, MLL-ELL, MLL-EP300, MLL-EPS15, MLL-FNBP1, MLL-FOXO3A, MLL-GMPS, MLL-GPHN, MLL-MLLT1, MLL-MLLT11, MLL-MLLT3, MLL-MLLT6, MLL-MYO1F, MLL-PICALM, MLL-SEPT2, MLL-SEPT6, MLL-SORBS2, MYST3-SORBS2, MYST-CREBBP, NPM1-MLF1, NUP98-HOXA13, PRDM16-EVI1, RABEP1-PDGFRB, RUNX1-EVI1, RUNX1-MDS1, RUNX1-RPL22, RUNX1-RUNX1T1, RUNX1-SH3D19, RUNX1-USP42, RUNX1-YTHDF2, RUNX1-ZNF687 or TAF15-ZNF-384, be selected from CCND1-FSTL3 for lymphocytic leukemia (CLL), and increase and be selected from FLIP1-PDGFRA, FLT3-ETV6, KIAA1509-PDGFRA, PDE4DIP-PDGFRB, NIN-PDGFRB, TP53BP1-PDGFRB or TPM3-PDGFRB for eosinophilia/chronic eosinophilic.

One or more biomarkers of CLL can also comprise following rise or the miRNA that cross to express in one or more, for example miR-23b, miR-24-1, miR-146, miR-155, miR-195, miR-221, miR-331, miR-29a, miR-195, miR-34a or miR-29c; And following downward or express one or more in not enough miR, for example miR-15a, miR-16-1, miR-29 or miR-223; Or their any combination.

One or more biomarkers of ALL can also comprise following rise or the miRNA that cross to express in one or more, for example miR-128b, miR-204, miR-218, miR-331, miR-181b-1, miR-17-92 or their any combination.

B cell lymphocytic leukemia (B-CLL)

B-CLL specific biological mark from vesica (for example can comprise one or more, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) miR that cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Figure 26, and can be for setting up the B-CLL specific biological marking.For example, the described biological marking can comprise that one or more cross the miR of expression, such as but not limited to miR-183-prec, miR-190, miR-24-1-prec, miR-33, miR-19a, miR-140, miR-123, miR-10b, miR-15b-prec, miR-92-1, miR-188, miR-154, miR-217, miR-101, miR-141-prec, miR-153-prec, miR-196-2, miR-134, miR-141, miR-132, miR-192 or miR-181b-prec or their any combination.

The described biological marking can also comprise that one or more express not enough miR, such as but not limited to miR-213, miR-220 or their any combination.One or more mRNA that can analyze can include but not limited to ZAP70, AdipoR1 or their any combination, and can be as the specific biological mark of the B-CLL from vesica.The biomarker sudden change of the B-CLL that can be assessed in vesica includes but not limited to the sudden change of IGHV, P53, ATM; Perhaps any combination of the specific sudden change of B-CLL.

The present invention also provides the vesica separated, and it comprises one or more B-CLL specific biological mark, for example BCL3-MYC, MYC-BTG1, BCL7A-MYC, BRWD3-ARHGAP20 or BTG1-MYC; Perhaps listed those in Figure 26.The composition of the vesica that comprises separation also is provided.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more B-CLL specific biological mark, for example BCL3-MYC, MYC-BTG1, BCL7A-MYC, BRWD3-ARHGAP20 or BTG1-MYC; Perhaps listed those in Figure 26.Described composition can comprise the vesica colony of obvious enrichment, wherein said vesica colony is for B-CLL specificity vesica or comprise one or more B-CLL specific biological marks, for example BCL3-MYC, MYC-BTG1, BCL7A-MYC, BRWD3-ARHGAP20 or BTG1-MYC, or in Figure 26, the vesica of those listed biomarkers is homogeneous basically.

For characterizing B-CLL, can also detect one or more B-CLL specific biological marks (for example in BCL3-MYC, MYC-BTG1, BCL7A-MYC, BRWD3-ARHGAP20 or BTG1-MYC or Figure 26 listed those) by one or more systems disclosed herein.For example, detection system can comprise one or more probes one or more B-CLL specific biological marks (for example those listed biomarkers in BCL3-MYC, MYC-BTG1, BCL7A-MYC, BRWD3-ARHGAP20 or BTG 1-MYC or Figure 26) with one or more vesicas in the detection of biological sample.

The B-cell lymphoma

B-cell lymphoma specific biological mark from vesica (for example can comprise one or more, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) miR that cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Figure 27, and can be for setting up the B-cell lymphoma specific biological marking.For example, the described biological marking can comprise that one or more cross the miR of expression, such as but not limited to miR-17-92 polycistron, miR-155, miR-210 or miR-21, miR-19a, miR-92, miR-142 miR-155, miR-221 miR-17-92, miR-21, miR-191, miR-205 or their any combination.In addition, can include but not limited to U50 as the snoRNA of the exosome biomarker of B-cell lymphoma.

The present invention also provides the vesica separated, and it comprises one or more B-cell lymphoma specific biological marks, for example listed in Figure 27.The composition of the vesica that comprises separation also is provided.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more B-cell lymphoma specific biological marks, for example listed in Figure 27.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony for example, is homogeneous basically for B-cell lymphoma specificity vesica or the vesica that comprises one or more B-cell lymphoma specific biological marks (listed in Figure 27).

For characterizing the B-cell lymphoma, can also detect one or more B-cell lymphoma specific biological marks (for example listed in Figure 27) by one or more systems disclosed herein.For example, detection system can comprise one or more probes one or more B-cell lymphoma specific biological marks (for example listed in Figure 27) with one or more vesicas in the detection of biological sample.

Diffuse large B cell lymphoma (DLBCL)

DLBCL specific biological mark from vesica (for example can comprise one or more, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) miR that cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Figure 28, and can be for setting up the DLBCL specific biological marking.For example, the described biological marking can comprise that one or more cross the miR of expression, such as but not limited to miR-17-92, miR-155, miR-210 or miR-21 or their any combination.One or more mRNA that can analyze can include but not limited to A-myb, LMO2, JNK3, CD10, bcl-6, Cyclin D2, IRF4, Flip, CD44 or their any combination, and can be as the specific biological mark of the DLBCL from vesica.

The present invention also provides the vesica separated, and it comprises one or more DLBCL specific biological mark, for example CITTA-BCL6, CLTC-ALK, IL21R-BCL6, PIM1-BCL6, TFCR-BCL6, IKZF1-BCL6 or SEC31A-ALK; Perhaps listed those biomarkers in Figure 28.The composition of the vesica that comprises separation also is provided.Therefore, in some embodiments, described composition comprises vesica colony, this colony comprises one or more DLBCL specific biological marks, for example listed those biomarkers in CITTA-BCL6, CLTC-ALK, IL21R-BCL6, PIM1-BCL6, TFCR-BCL6, IKZF1-BCL6 or SEC31A-ALK or Figure 28.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony for example, is homogeneous basically for DLBCL specificity vesica or the vesica that comprises one or more DLBCL specific biological marks (in CITTA-BCL6, CLTC-ALK, IL21R-BCL6, PIM1-BCL6, TFCR-BCL6, IKZF1-BCL6 or SEC31A-ALK or Figure 28 listed those biomarkers).

For characterizing DLBCL, can also detect one or more DLBCL specific biological marks (for example in CITTA-BCL6, CLTC-ALK, IL21R-BCL6, PIM1-BCL6, TFCR-BCL6, IKZF1-BCL6 or SEC31A-ALK or Figure 28 listed those biomarkers) by one or more systems disclosed herein.For example, detection system can comprise that one or more probes for example, with one or more DLBCL specific biological marks of one or more vesicas in the detection of biological sample (in CITTA-BCL6, CLTC-ALK, IL21R-BCL6, PIM1-BCL6, TFCR-BCL6, IKZF1-BCL6 or SEC31A-ALK or Figure 28 listed those).

Burkitt's lymphoma

Burkitt's lymphoma specific biological mark from vesica (for example can comprise one or more, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) miR that cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Figure 29, and can be for setting up the burkitt's lymphoma specific biological marking.For example, the described biological marking can also comprise that one or more express not enough miR, such as but not limited to pri-miR-155 or their any combination.One or more mRNA that can analyze can include but not limited to MYC, TERT, NS, NP, MAZ, RCF3, BYSL, IDE3, CDC7, TCL1A, AUTS2, MYBL1, BMP7, ITPR3, CDC2, BACK2, TTK, MME, ALOX5 or TOP1 or their any combination, and can be as the specific biological mark of the burkitt's lymphoma from vesica.The protein that can be assessed in vesica, part or peptide can include but not limited to BCL6, KI-67 or their any combination.

The present invention also provides the vesica separated, and it comprises one or more burkitt's lymphoma specific biological mark, for example IGH-MYC, LCP1-BCL6; Perhaps listed those biomarkers in Figure 29.The composition of the vesica that comprises separation also is provided.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more burkitt's lymphoma specific biological marks (for example in IGH-MYC, LCP1-BCL6 or Figure 29 listed those biomarkers).Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony's burkitt's lymphoma specificity vesica or the vesica that comprises one or more burkitt's lymphoma specific biological marks (for example in IGH-MYC, LCP1-BCL6 or Figure 29 listed those biomarkers) are homogeneous basically.

For characterizing burkitt's lymphoma, can also detect for example, in one or more burkitt's lymphoma specific biological marks (IGH-MYC, LCP1-BCL6) or Figure 29 those listed biomarkers by one or more systems disclosed herein.For example, detection system can comprise that one or more probes for example, with one or more burkitt's lymphoma specific biological marks of one or more vesicas in the detection of biological sample (in IGH-MYC, LCP1-BCL6 or Figure 29 listed those biomarkers).

Hepatocellular carcinoma

Hepatocellular carcinoma specific biological mark from vesica (for example can comprise one or more, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) miR that cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Figure 30, and can be for setting up the hepatocellular carcinoma specific biological marking.For example, the described biological marking can comprise that one or more cross the miR of expression, such as but not limited to miR-221.The described biological marking also can comprise that one or more express not enough miR, such as but not limited to let-7a-1, let-7a-2, let-7a-3, let-7b, let-7c, let-7d, let-7e, let-7f-2, let-fg, miR-122a, miR-124a-2, miR-130a, miR-132, miR-136, miR-141, miR-142, miR-143, miR-145, miR-146, miR-150, miR-155 (BIC), miR-181a-1, miR-181a-2, miR-181c, miR-195, miR-199a-1-5p, miR-199a-2-5p, miR-199b, miR-200b, miR-214, miR-223 or pre-miR-594 or their any combination.One or more mRNA that can analyze can include but not limited to FAT10.

One or more biomarkers of the biological marking can also be for characterizing the hepatitis C virus related Hepatocellular Carcinoma.Described one or more biomarkers can be miRNA, for example cross to express or express not enough miRNA.For example, raising or cross the miRNA expressed can be miR-122, miR-100 or miR-10a, and the miRNA lowered can be miR-198 or miR-145.

The present invention also provides the vesica separated, and it comprises one or more hepatocellular carcinoma specific biological marks, for example listed for hepatocellular carcinoma in Figure 30 and Fig. 1.The composition of the vesica that comprises separation also is provided.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more hepatocellular carcinoma specific biological marks, for example listed for hepatocellular carcinoma in Figure 30 and Fig. 1.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony for example, is homogeneous basically for hepatocellular carcinoma specificity vesica or the vesica that comprises one or more hepatocellular carcinoma specific biological marks (listed for hepatocellular carcinoma in Figure 30 and Fig. 1).

For characterizing hepatocellular carcinoma, can also detect one or more hepatocellular carcinoma specific biological marks (for example listed for hepatocellular carcinoma in Figure 30 and Fig. 1) by one or more systems disclosed herein.For example, detection system can comprise one or more probes one or more hepatocellular carcinoma specific biological marks (for example listed for hepatocellular carcinoma in Figure 30 and Fig. 1) with one or more vesicas in the detection of biological sample.

Cervical cancer

Cervical cancer specific biological mark from vesica (for example can comprise one or more, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) miR that cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Figure 31, and can be for setting up the cervical cancer specific biological marking.For example, one or more mRNA that can analyze can include but not limited to HPV E6, HPV E7 or p53 or their any combination, and can be as the specific biological mark of the cervical cancer from vesica.

The present invention also provides the vesica separated, and it comprises one or more cervical cancer specific biological marks, for example listed for cervical cancer in Figure 31 and Fig. 1.The composition of the vesica that comprises separation also is provided in addition.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more cervical cancer specific biological marks, for example listed for cervical cancer in Figure 31 and Fig. 1.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony for example, is homogeneous basically for cervical cancer specificity vesica or the vesica that comprises one or more cervical cancer specific biological marks (in Figure 31 and Fig. 1 for cervical cancer listed biomarker).

For characterizing cervical cancer, can also detect one or more cervical cancer specific biological marks (for example listed for cervical cancer in Figure 31 and Fig. 1) by one or more systems disclosed herein.For example, detection system can comprise one or more probes one or more cervical cancer specific biological marks (for example listed for cervical cancer in Figure 31 and Fig. 1) with one or more vesicas in the detection of biological sample.

Carcinoma of endometrium

Carcinoma of endometrium specific biological mark from vesica (for example can comprise one or more, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) miR that cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Figure 32, and can be for setting up the carcinoma of endometrium specific biological marking.For example, the described biological marking can comprise the miR that one or more cross expression, such as but not limited to miR-185, miR-106a, miR-181a, miR-210, miR-423, miR-103, miR-107 or let-7c or their any combination.The described biological marking can also comprise one or more and express not enough miR, such as but not limited to miR-7i, miR-221, miR-193, miR-152 or miR-30c or their any combination.

The biomarker sudden change of the carcinoma of endometrium that can be assessed in vesica includes but not limited to the sudden change of PTEN, K-RAS, B-catenin, p53, Her2/neu; Perhaps any combination of the specific sudden change of carcinoma of endometrium.The protein that can be assessed in vesica, part or peptide can include but not limited to NLRP7, α V β 6 integrins or their any combination.

The present invention also provides the vesica separated, and it comprises one or more carcinoma of endometrium specific biological marks, for example listed for carcinoma of endometrium in Figure 32 and Fig. 1.The composition of the vesica that comprises separation also is provided.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more carcinoma of endometrium specific biological marks, for example listed for carcinoma of endometrium in Figure 32 and Fig. 1.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony for example, is homogeneous basically for carcinoma of endometrium specificity vesica or the vesica that comprises one or more carcinoma of endometrium specific biological marks (listed for carcinoma of endometrium in Figure 32 and Fig. 1).

For characterizing carcinoma of endometrium, can also detect one or more carcinoma of endometrium specific biological marks (for example listed for carcinoma of endometrium in Figure 32 and Fig. 1) by one or more systems disclosed herein.For example, detection system can comprise one or more probes one or more carcinoma of endometrium specific biological marks (for example listed for carcinoma of endometrium in Figure 32 and Fig. 1) with one or more vesicas in the detection of biological sample.

Head and neck cancer

Head and neck cancer specific biological mark from vesica (for example can comprise one or more, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) miR that cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Figure 33, and can be for setting up the specific biological marking of head and neck cancer.For example, the described biological marking can comprise the miR that one or more cross expression, such as but not limited to miR-21, let-7, miR-18, miR-29c, miR-142-3p, miR-155, miR-146b, miR-205 or miR-21 or their any combination.The described biological marking can also comprise one or more and express not enough miR, such as but not limited to miR-494.One or more mRNA that can analyze include but not limited to HPV E6, HPV E7, p53, IL-8, SAT, H3FA3 or EGFR or their any combination, and can be as the specific biological mark of the head and neck cancer from vesica.

The biomarker sudden change of the head and neck cancer that can be assessed in vesica includes but not limited to the sudden change of GSTM1, GSTT1, GSTP1, OGG1, XRCC1, XPD, RAD51, EGFR, p53; Perhaps any combination of the specific mutant of head and neck cancer.The protein that can be assessed in vesica, part or peptide can include but not limited to EGFR, EphB4 or EphB2 or their any combination.

The present invention also provides the vesica separated, and it comprises one or more head and neck cancer specific biological marks, for example CHCHD7-PLAG1, CTNNB 1-PLAG1, FHIT-HMGA2, HMGA2-NFIB, LIFR-PLAG1 or TCEA1-PLAG1; Perhaps in Figure 33 and Fig. 1 for head and neck cancer listed those biomarkers.The composition of the vesica that comprises separation also is provided.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more head and neck cancer specific biological mark, for example CHCHD7-PLAG1, CTNNB1-PLAG1, FHIT-HMGA2, HMGA2-NFIB, LIFR-PLAG1 or TCEA1-PLAG1; Perhaps in Figure 33 and Fig. 1 for head and neck cancer listed those biomarkers.Described composition can comprise the vesica colony of obvious enrichment, wherein said vesica colony is for head and neck cancer specificity vesica or comprise one or more head and neck cancer specific biological mark, for example CHCHD7-PLAG1, CTNNB1-PLAG1, FHIT-HMGA2, HMGA2-NFIB, LIFR-PLAG1 or TCEA1-PLAG1; Perhaps in Figure 33 and Fig. 1, for head and neck cancer, the vesica of those listed biomarkers is homogeneous basically.

For characterizing head and neck cancer, can also detect one or more head and neck cancer specific biological marks (for example in Figure 33 and Fig. 1 for head and neck cancer listed those) by one or more systems disclosed herein.For example, detection system can comprise that one or more probes for example, with one or more head and neck cancer specific biological marks of one or more vesicas in the detection of biological sample (CHCHD7-PLAG1, CTNNB1-PLAG1, FHIT-HMGA2, HMGA2-NFIB, LIFR-PLAG1 or TCEA1-PLAG1; Perhaps in Figure 33 and Fig. 1 for head and neck cancer listed those biomarkers).

Inflammatory bowel (IBD)

IBD specific biological mark from vesica (for example can comprise one or more, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) miR that cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Figure 34, and can be for setting up the IBD specific biological marking.One or more mRNA that can analyze can include but not limited to trypsinogen IV, SERT or their any combination, and can be as the specific biological mark of the IBD from vesica.

The biomarker sudden change of the IBD that can be assessed in vesica can include but not limited to the sudden change of CARD15; Perhaps any combination of the specific sudden change of IBD.The protein that can be assessed in vesica, part or peptide can include but not limited to II-16, II-1 β, II-12, TNF-α, interferon-gamma, II-6, Rantes, MCP-1, phylaxin or 5-HT or their any combination.

The present invention also provides the vesica separated, and it comprises one or more IBD specific biological marks, for example listed for IBD in Figure 34 and Fig. 1.The composition of the vesica that comprises separation also is provided in addition.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more IBD specific biological marks, for example listed for IBD in Figure 34 and Fig. 1.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony for example, is homogeneous basically for IBD specificity vesica or the vesica that comprises one or more IBD specific biological marks (listed for IBD in Figure 34 and Fig. 1).

For characterizing IBD, can also detect one or more IBD specific biological marks (for example listed for IBD in Figure 34 and Fig. 1) by one or more systems disclosed herein.For example, detection system can comprise one or more probes one or more IBD specific biological marks (for example listed for IBD in Figure 34 and Fig. 1) with one or more vesicas in the detection of biological sample.

Diabetes

Diabetes specific biological mark from vesica (for example can comprise one or more, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) miR that cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Figure 35, and can be for setting up the diabetes specific biological marking.For example, one or more mRNA that can analyze can include but not limited to Il-8, CTSS, ITGB2, HLA-DRA, CD53, PLAG27 or MMP9 or their any combination, and can be as the specific biological mark of the diabetes from vesica.The protein that can be assessed in vesica, part or peptide can include but not limited to RBP4.

The present invention also provides the vesica separated, and it comprises one or more diabetes specific biological marks, for example listed for diabetes in Figure 35 and Fig. 1.The composition of the vesica that comprises separation also is provided in addition.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more diabetes specific biological marks, for example in Figure 35 and Fig. 1 for diabetes listed biomarker.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony for example, is homogeneous basically for diabetes specificity vesica or the vesica that comprises one or more diabetes specific biological marks (listed for diabetes in Figure 35 and Fig. 1).

For characterizing diabetes, can also detect one or more diabetes specific biological marks (for example listed for diabetes in Figure 35 and Fig. 1) by one or more systems disclosed herein.For example, detection system can comprise one or more probes one or more diabetes specific biological marks (for example listed for diabetes in Figure 35 and Fig. 1) with one or more vesicas in the detection of biological sample.

Barrett esophagus

Barrett esophagus specific biological mark from vesica (for example can comprise one or more, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) miR that cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Figure 36, and can be for setting up the Barrett esophagus specific biological marking.For example, the described biological marking can comprise the miR that one or more cross expression, such as but not limited to miR-21, miR-143, miR-145, miR-194 or miR-215 or their any combination.One or more mRNA that can analyze include but not limited to S100A2, S100A4 or their any combination, and can be as the specific biological mark of the Barrett esophagus from vesica.

The biomarker sudden change of the Barrett esophagus that can be assessed in vesica can include but not limited to the sudden change of p53; Perhaps any combination of Barrett esophagus specific mutant.The protein that can be assessed in vesica, part or peptide can include but not limited to p53, MUC1, MUC2 or their any combination.

The present invention also provides the vesica separated, and it comprises one or more Barrett esophagus specific biological marks, for example listed for Barrett esophagus in Figure 36 and Fig. 1.The composition of the vesica that comprises separation also is provided.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more Barrett esophagus specific biological marks, for example listed for Barrett esophagus in Figure 36 and Fig. 1.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony for example, is homogeneous basically for Barrett esophagus specificity vesica or the vesica that comprises one or more Barrett esophagus specific biological marks (listed for Barrett esophagus in Figure 36 and Fig. 1).

For characterizing Barrett esophagus, can also detect by one or more systems disclosed herein the specific biological mark (for example in Figure 36 and Fig. 1 for Barrett esophagus listed biomarker) of one or more Barrett esophaguses.For example, detection system can comprise one or more probes one or more Barrett esophagus specific biological marks (for example listed for Barrett esophagus in Figure 36 and Fig. 1) with one or more vesicas in the detection of biological sample.

Fibromyalgia

Fibromyalgia specific biological mark from vesica (for example can comprise one or more, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) miR that cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Figure 37, and can be for setting up the fibromyalgia specific biological marking.One or more mRNA that can analyze include but not limited to NR2D, and it can be as the specific biological mark of the fibromyalgia from vesica.

The present invention also provides the vesica separated, and it comprises one or more fibromyalgia specific biological marks, for example in Figure 37 and Fig. 1 for fibromyalgia listed biomarker.The composition of the vesica that comprises separation also is provided.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more fibromyalgia specific biological marks, for example in Figure 37 and Fig. 1 for fibromyalgia listed biomarker.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony for example, is homogeneous basically for fibromyalgia specificity vesica or the vesica that comprises one or more fibromyalgia specific biological marks (in Figure 37 and Fig. 1 for fibromyalgia listed biomarker).

For characterizing fibromyalgia, can also detect by one or more systems disclosed herein the specific biological mark (for example in Figure 37 and Fig. 1 for fibromyalgia listed biomarker) of one or more fibromyalgias.For example, detection system can comprise that one or more probes for example, with one or more fibromyalgia specific biological marks of one or more vesicas in the detection of biological sample (in Figure 37 and Fig. 1 for fibromyalgia listed biomarker).

Apoplexy

Apoplexy specific biological mark from vesica (for example can comprise one or more, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) miR that cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Figure 38, and can be for setting up the apoplexy specific biological marking.For example; one or more mRNA that can analyze can include but not limited to MMP9, S100-P, S100A12, S100A9, factor V, arginase I, CA-IV, monocarboxylate transporter, ets-2, EIF2 α, protein 4, N-formyl peptide receptor, rnase 2, N-acetyl-neuraminate acetone lyase, BCL-6 or glycogen phosphorylase or their any combination that cytoskeleton is relevant, and can be as the specific biological mark of the apoplexy from vesica.

The present invention also provides the vesica separated, and it comprises one or more apoplexy specific biological marks, for example listed for apoplexy in Figure 38 and Fig. 1.The composition of the vesica that comprises separation also is provided in addition.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more apoplexy specific biological marks, for example in Figure 38 and Fig. 1 for apoplexy listed biomarker.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony for example, is homogeneous basically for apoplexy specificity vesica or the vesica that comprises one or more apoplexy specific biological marks (in Figure 38 and Fig. 1 for apoplexy listed biomarker).

For characterizing apoplexy, can also detect by one or more systems disclosed herein the specific biological mark (for example in Figure 38 and Fig. 1 for apoplexy listed biomarker) of one or more apoplexy.For example, detection system can comprise that one or more probes for example, with one or more apoplexy specific biological marks of one or more vesicas in the detection of biological sample (in Figure 38 and Fig. 1 for apoplexy listed biomarker).

Multiple sclerosis (MS)

MS specific biological mark from vesica (for example can comprise one or more, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) miR that cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Figure 39, and can be for setting up the MS specific biological marking.For example, one or more mRNA that can analyze can include but not limited to IL-6, IL-17, PAR-3, IL-17, T1/ST2, JunD, 5-LO, LTA4H, MBP, PLP or alpha-beta crystallin or their any combination, and can be as the specific biological mark of the MS from vesica.

The present invention also provides the vesica separated, and it comprises one or more MS specific biological marks, for example listed for MS in Figure 39 and Fig. 1.The composition of the vesica that comprises separation also is provided in addition.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more MS specific biological marks, for example in Figure 39 and Fig. 1 for MS listed biomarker.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony for example, is homogeneous basically for MS specificity vesica or the vesica that comprises one or more MS specific biological marks (in Figure 39 and Fig. 1 for MS listed biomarker).

For characterizing MS, can also detect by one or more systems disclosed herein the specific biological mark (for example in Figure 39 and Fig. 1 for MS listed biomarker) of one or more MS.For example, detection system can comprise that one or more probes for example, with one or more MS specific biological marks of one or more vesicas in the detection of biological sample (in Figure 39 and Fig. 1 for MS listed biomarker).

Parkinson's disease

Parkinson's disease specific biological mark from vesica (for example can comprise one or more, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) miR that cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Figure 40, and can be for setting up the Parkinson's disease specific biological marking.For example, the described biological marking can include but not limited to that one or more express not enough miR, for example miR-133b.One or more mRNA that can analyze can include but not limited to Nurr1, BDNF, TrkB, gstm1 or S100 β or their any combination, and can be as the parkinsonian specific biological mark from vesica.

The parkinsonian biomarker sudden change that can be assessed in vesica can include but not limited to the sudden change of FGF20, alpha-synapse nucleoprotein, FGF20, NDUFV2, FGF2, CALB1, B2M; Perhaps any combination of parkinsonian specific mutant.The protein that can be assessed in vesica, part or peptide can include but not limited to apo-H, ceruloplasmin, BDNF, IL-8, B2M, apoAII, tau, A β 1-42, DJ-1 or their any combination.

The present invention also provides the vesica separated, and it comprises one or more Parkinson's disease specific biological marks, for example listed for Parkinson's disease in Figure 40 and Fig. 1.The composition of the vesica that comprises separation also is provided in addition.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more Parkinson's disease specific biological marks, for example in Figure 40 and Fig. 1 for Parkinson's disease listed biomarker.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony for example, is homogeneous basically for Parkinson disease-specific vesica or the vesica that comprises one or more Parkinson's disease specific biological marks (in Figure 40 and Fig. 1 for Parkinson's disease listed biomarker).

For characterizing Parkinson's disease, can also detect one or more parkinsonian specific biological marks (for example in Figure 40 and Fig. 1 for Parkinson's disease listed biomarker) by one or more systems disclosed herein.For example, detection system can comprise that one or more probes for example, with one or more Parkinson's disease specific biological marks of one or more vesicas in the detection of biological sample (in Figure 40 and Fig. 1 for Parkinson's disease listed biomarker).

Rheumatosis

Rheumatosis specific biological mark from vesica (for example can comprise one or more, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) miR that cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Figure 41, and can be for setting up the rheumatosis specific biological marking.For example, the described biological marking can comprise that one or more express not enough miR, such as but not limited to miR-146a, miR-155, miR-132, miR-16 or miR-181 or their any combination.One or more mRNA that can analyze can include but not limited to HOXD10, HOXD11, HOXD13, CCL8, LIM homology frame 2 or CENP-E or their any combination, and can be as the rheumatismal specific biological mark from vesica.The protein that can be assessed in vesica, part or peptide can include but not limited to TNF α.

The present invention also provides the vesica separated, and it comprises one or more rheumatosis specific biological marks, for example in Figure 41 and Fig. 1 for rheumatosis listed biomarker.The composition of the vesica that comprises separation also is provided in addition.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more rheumatosis specific biological marks, for example in Figure 41 and Fig. 1 for rheumatosis listed biomarker.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony for example, is homogeneous basically for rheumatosis specificity vesica or the vesica that comprises one or more rheumatosis specific biological marks (in Figure 41 and Fig. 1 for rheumatosis listed biomarker).

For characterizing rheumatosis, can also detect one or more rheumatosis specific biological marks (for example in Figure 41 and Fig. 1 for rheumatosis listed biomarker) by one or more systems disclosed herein.For example, detection system can comprise that one or more probes for example, with one or more rheumatosis specific biological marks of one or more vesicas in the detection of biological sample (in Figure 41 and Fig. 1 for rheumatosis listed biomarker).

Alzheimer's disease

Alzheimer's disease specific biological mark from vesica (for example can comprise one or more, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) miR that cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Figure 42, and can be for setting up the alzheimer's disease specific biological marking.For example, the described biological marking can also comprise that one or more express not enough miR, for example miR-107, miR-29a, miR-29b-1 or miR-9 or their any combination.The described biological marking can also comprise that one or more cross the miR of expression, for example miR-128 or their any combination.

One or more mRNA that can analyze can include but not limited to HIF-1 α, BACE1, Reelin, CHRNA7 or 3Rtau/4Rtau or their any combination, and can be as the specific biological mark of the alzheimer's disease from vesica.

The biomarker sudden change of the alzheimer's disease that can be assessed in vesica can include but not limited to the sudden change of APP, early ageing albumen 1, early ageing albumen 2, APOE4; Perhaps any combination of the specific mutant of alzheimer's disease.The protein that can be assessed in vesica, part or peptide can include but not limited to Cystatin C, starch albumen β, C3a, t-Tau, complement factor H or α-2-macroglobulin or their any combination of BACE1, Reelin, Cystatin C, brachymemma.

The present invention also provides the vesica separated, and it comprises one or more alzheimer's disease specific biological marks, for example in Figure 42 and Fig. 1 for alzheimer's disease listed biomarker.The composition of the vesica that comprises separation also is provided in addition.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more alzheimer's disease specific biological marks, for example in Figure 42 and Fig. 1 for alzheimer's disease listed biomarker.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony for example, is homogeneous basically for alzheimer's disease specificity vesica or the vesica that comprises one or more alzheimer's disease specific biological marks (in Figure 42 and Fig. 1 for alzheimer's disease listed biomarker).

For characterizing alzheimer's disease, can also detect one or more alzheimer's disease specific biological marks (for example in Figure 42 and Fig. 1 for alzheimer's disease listed biomarker) by one or more systems disclosed herein.For example, detection system can comprise that one or more probes for example, with one or more alzheimer's disease specific biological marks of one or more vesicas in the detection of biological sample (in Figure 42 and Fig. 1 for alzheimer's disease listed biomarker).

Prion disease

Prion-specific biomarker from vesica (for example can comprise one or more, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) miR that cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Figure 43, and can be for setting up the biological marking of prion-specific.For example, one or more mRNA that can analyze can include but not limited to amyloid B4, App, IL-1R1 or SOD1 or their any combination, and can be as the prion-specific biomarker from vesica.The protein that can be assessed in vesica, part or peptide can include but not limited to PrP (c), 14-3-3, NSE, S-100, Tau, AQP-4 or their any combination.

The present invention also provides the vesica separated, and it comprises one or more prion-specific biomarkers, for example in Figure 43 and Fig. 1 for prion disease listed biomarker.The composition of the vesica that comprises separation also is provided in addition.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more prion-specific biomarkers, for example in Figure 43 and Fig. 1 for prion disease listed biomarker.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony for example, is homogeneous basically for prion disease specificity vesica or the vesica that comprises one or more prion disease specific biological marks (in Figure 43 and Fig. 1 for prion disease listed biomarker).

For characterizing prion disease, can also detect one or more prion disease specific biological marks (for example in Figure 43 and Fig. 1 for prion disease listed biomarker) by one or more systems disclosed herein.For example, detection system can comprise that one or more probes for example, with one or more prion disease specific biological marks of one or more vesicas in the detection of biological sample (in Figure 43 and Fig. 1 for prion disease listed biomarker).

Sepsis

Sepsis specific biological mark from vesica (for example can comprise one or more, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) miR that cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Figure 44, and can be for setting up the Sepsis specific biological marking.For example, one or more mRNA that can analyze can include but not limited to 15-hydroxyl-PG desaturase (making progress), LAIR1 (making progress), NFKB1A (making progress), TLR2, PGLYPR1, TLR4, MD2, TLR5, IFNAR2, IRAK2, IRAK3, IRAK4, PI3K, PI3KCB, MAP2K6, MAPK14, NFKB1A, NFKB1, IL1R1, MAP2K1IP1, MKNK1, FAS, CASP4, GADD45B, SOCS3, TNFSF10, TNFSF13B, OSM, HGF or IL18R1 or their any combination, and can be as the pyemic specific biological mark from vesica.

The present invention also provides the vesica separated, and it comprises one or more Sepsis specific biological marks, and for example Figure 44 is listed.The composition of the vesica that comprises separation also is provided in addition.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more Sepsis specific biological marks, for example listed in Figure 44.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony for example, is homogeneous basically for Sepsis specificity vesica or the vesica that comprises one or more Sepsis specific biological marks (in Figure 44 listed biomarker).

For characterizing Sepsis, can also detect one or more Sepsis specific biological marks (for example in Figure 44 listed biomarker) by one or more systems disclosed herein.For example, detection system can comprise that one or more probes for example, with one or more Sepsis specific biological marks of one or more vesicas in the detection of biological sample (in Figure 44 listed biomarker).

Chronic neuropathic pain model

Chronic neuropathic pain model (CNP) specific biological mark from vesica (for example can comprise one or more, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) miR that cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Figure 45, and can be for setting up the CNP specific biological marking.For example, one or more mRNA that can analyze can include but not limited to ICAM-1 (rodent), CGRP (rodent), TIMP-1 (rodent), CLR-1 (rodent), HSP-27 (rodent), FABP (rodent) or Apolipoprotein D (rodent) or their any combination, and can be as the specific biological mark of the CNP from vesica.The protein that can be assessed in vesica, part or peptide can include but not limited to chemokine, Chemokine Receptors (CCR2/4) or their any combination.

The present invention also provides the vesica separated, and it comprises one or more chronic neuropathic pain model specific biological marks, for example in Figure 45 and Fig. 1 for chronic neuropathic pain model listed biomarker.The composition of the vesica that comprises separation also is provided in addition.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more chronic neuropathic pain model specific biological marks, for example in Figure 45 and Fig. 1 for chronic neuropathic pain model listed biomarker.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony for example, is homogeneous basically for chronic neuropathic pain model specificity vesica or the vesica that comprises one or more chronic neuropathic pain model specific biological marks (in Figure 45 and Fig. 1 for chronic neuropathic pain model listed biomarker).

For characterizing chronic neuropathic pain model, can also detect one or more chronic neuropathic pain model specific biological marks (for example in Figure 45 and Fig. 1 for chronic neuropathic pain model listed biomarker) by one or more systems disclosed herein.For example, detection system can comprise that one or more probes for example, with one or more chronic neuropathic pain model specific biological marks of one or more vesicas in the detection of biological sample (in Figure 45 and Fig. 1 for chronic neuropathic pain model listed biomarker).

Peripheral nerve pathologic pain

Peripheral nerve pathologic pain (PNP) specific biological mark from vesica (for example can comprise one or more, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) miR that cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Figure 46, and can be for setting up the PNP specific biological marking.For example, the protein that can be assessed in vesica, part or peptide can include but not limited to OX42, ED9 or their any combination.

The present invention also provides the vesica separated, and it comprises one or more peripheral nerve pathologic pain specific biomarkers, for example in Figure 46 and Fig. 1 for the listed biomarker of peripheral nerve pathologic pain.The composition of the vesica that comprises separation also is provided.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more peripheral nerve pathologic pain specific biomarkers, for example in Figure 46 and Fig. 1 for the listed biomarker of peripheral nerve pathologic pain.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony for example, is homogeneous basically for peripheral nerve pathologic pain specific vesica or the vesica that comprises one or more peripheral nerve pathologic pain specific biomarkers (in Figure 46 and Fig. 1 for the listed biomarker of peripheral nerve pathologic pain).

For characterizing peripheral nerve pathologic pain, can also detect one or more peripheral nerve pathologic pain specific biomarkers (for example in Figure 46 and Fig. 1 for the listed biomarker of peripheral nerve pathologic pain) by one or more systems disclosed herein.For example, detection system can comprise that one or more probes for example, with one or more peripheral nerve pathologic pain specific biomarkers of one or more vesicas in the detection of biological sample (in Figure 46 and Fig. 1 for the listed biomarker of peripheral nerve pathologic pain).

Schizophrenia

Schizophrenia specific biological mark from vesica (for example can comprise one or more, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) miR that cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Figure 47, and can be for setting up the schizophrenia specific biological marking.For example, the described biological marking can comprise that one or more cross the miR of expression, such as but not limited to miR-181b.The described biological marking can also comprise that one or more express not enough miR, such as but not limited to miR-7, miR-24, miR-26b, miR-29b, miR-30b, miR-30e, miR-92 or miR-195 or their any combination.

One or more mRNA that can analyze can include but not limited to IFITM3, SERPINA3, GLS or ALDH7A1BASP1 or their any combination, and can be as the schizoid specific biological mark from vesica.The schizoid biomarker sudden change that can be assessed in vesica includes but not limited to the sudden change of DISC1, dysbindin, neuregulin-1, seratonin 2a acceptor, NURR1; Perhaps any combination of schizoid specific mutant.

The protein that can be assessed in vesica, part or peptide can include but not limited to ATP5B, ATP5H, ATP6V1B, DNM1, NDUFV2, NSF, PDHB or their any combination.

The present invention also provides the vesica separated, and it comprises one or more schizophrenia specific biological marks, for example in Figure 47 and Fig. 1 for schizophrenia listed biomarker.The composition of the vesica that comprises separation also is provided in addition.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more schizophrenia specific biological marks, for example in Figure 47 and Fig. 1 for schizophrenia listed biomarker.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony for example, is homogeneous basically for schizophrenia specificity vesica or the vesica that comprises one or more schizophrenia specific biological marks (in Figure 47 and Fig. 1 for schizophrenia listed biomarker).

For characterizing schizophrenia, can also detect one or more schizophrenia specific biological marks (for example in Figure 47 and Fig. 1 for schizophrenia listed biomarker) by one or more systems disclosed herein.For example, detection system can comprise that one or more probes for example, with one or more schizophrenia specific biological marks of one or more vesicas in the detection of biological sample (in Figure 47 and Fig. 1 for schizophrenia listed biomarker).

The biphasic or bipolar type disease

Biphasic or bipolar type disease specific biomarker from vesica (for example can comprise one or more, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) miR that cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Figure 48, and can be for setting up the biological marking of biphasic or bipolar type disease specific.For example, one or more mRNA that can analyze can include but not limited to FGF2, ALDH7A1, AGXT2L1, AQP4 or PCNT2 or their any combination, and can be as the specific biological mark of the biphasic or bipolar type disease from vesica.The biomarker sudden change of the biphasic or bipolar type disease that can be assessed in vesica includes but not limited to the sudden change of dysbindin, DAOA/G30, DISC1, neuregulin-1; Perhaps any combination of biphasic or bipolar type disease specific sudden change.

The present invention also provides the vesica separated, and it comprises one or more biphasic or bipolar type disease specific biomarkers, for example listed in Figure 48.The composition of the vesica that comprises separation also is provided.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more biphasic or bipolar type disease specific biomarkers, for example listed in Figure 48.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony for example, is homogeneous basically for biphasic or bipolar type disease specific vesica or the vesica that comprises one or more biphasic or bipolar type disease specific biomarkers (in Figure 48 listed biomarker).

For characterizing the biphasic or bipolar type disease, can also detect one or more biphasic or bipolar type disease specific biomarkers (for example in Figure 48 listed biomarker) by one or more systems disclosed herein.For example, detection system can comprise that one or more probes for example, with one or more biphasic or bipolar type disease specific biomarkers of one or more vesicas in the detection of biological sample (in Figure 48 listed biomarker).

Dysthymia disorders

Dysthymia disorders specific biological mark from vesica (for example can comprise one or more, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) miR that cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Figure 49, and can be for setting up the dysthymia disorders specific biological marking.For example, one or more mRNA that can analyze can include but not limited to FGFR1, FGFR2, FGFR3 or AQP4 or their any combination, and can be as the specific biological mark of the dysthymia disorders from vesica.

The present invention also provides the vesica separated, and it comprises one or more dysthymia disorders specific biological marks, for example listed in Figure 49.The composition of the vesica that comprises separation also is provided in addition.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more dysthymia disorders specific biological marks, for example listed biomarker in Figure 49.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony for example, is homogeneous basically for dysthymia disorders specificity vesica or the vesica that comprises one or more dysthymia disorders specific biological marks (in Figure 49 listed biomarker).

For characterizing dysthymia disorders, can also detect one or more dysthymia disorders specific biological marks (for example in Figure 49 listed biomarker) by one or more systems disclosed herein.For example, detection system can comprise that one or more probes for example, with one or more dysthymia disorders specific biological marks of one or more vesicas in the detection of biological sample (in Figure 49 listed biomarker).

Gastrointestinal stromal tumor (GIST)

GIST specific biological mark from vesica (for example can comprise one or more, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) miR that cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Figure 50, and can be for setting up the GIST specific biological marking.For example, one or more mRNA that can analyze can include but not limited to DOG-1, PKC-θ, KIT, GPR20, PRKCQ, KCNK3, KCNH2, SCG2, TNFRSF6B or CD34 or their any combination, and can be as the specific biological mark of the GIST from vesica.

The biomarker sudden change of the GIST that can be assessed in vesica can include but not limited to the sudden change of PKC-θ; Perhaps any combination of GIST specific mutant.The protein that can be assessed in vesica, part or peptide can include but not limited to PDGFRA, c-kit or their any combination.

The present invention also provides the vesica separated, and it comprises one or more GIST specific biological marks, for example in Figure 50 and Fig. 1 for GIST listed biomarker.The composition of the vesica that comprises separation also is provided in addition.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more GIST specific biological marks, for example in Figure 50 and Fig. 1 for GIST listed biomarker.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony for example, is homogeneous basically for GIST specificity vesica or the vesica that comprises one or more GIST specific biological marks (in Figure 50 and Fig. 1 for GIST listed biomarker).

For characterizing GIST, can also detect one or more GIST specific biological marks (for example in Figure 50 and Fig. 1 for GIST listed biomarker) by one or more systems disclosed herein.For example, detection system can comprise that one or more probes for example, with one or more GIST specific biological marks of one or more vesicas in the detection of biological sample (in Figure 50 and Fig. 1 for GIST listed biomarker).

Renal cell carcinoma

Renal cell carcinoma specific biological mark from vesica (for example can comprise one or more, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) miR that cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Figure 51, and can be for setting up the renal cell carcinoma specific biological marking.For example, the described biological marking can also comprise that one or more express not enough miR, such as but not limited to miR-141, miR-200c or their any combination.Described one or more rises or the miRNA excessively expressed can be miR-28, miR-185, miR-27, miR-let-7f-2 or their any combination.

One or more mRNA that can analyze can include but not limited to laminin receptor 1, betaig-h3, half lactadherin-1, the a-2 macroglobulin, IMA-ADF-001, angiogenesis promoting protein factor 2, caldesmon 1, the II class MHC-invariant chain (CD74) of being correlated with, collagen protein IV-a1, complement component, complement component 3, Cytochrome P450, IIJ subfamily polypeptide 2, the δ sleep inducing peptide, Fc g receptor II Ia (CD16), HLA-B, HLA-DRa, HLA-DRb, HLA-SB, the transmembrane protein 3 that IFN-induces, the transmembrane protein 1 that IFN-induces or lysyloxidase or their any combination, and can be as the renal cell carcinoma specific biological mark that derives from vesica.

The biomarker sudden change of the renal cell carcinoma that can be assessed in vesica includes but not limited to the sudden change of VHL; Perhaps any combination of renal cell carcinoma specific mutant.

The protein that can be assessed in vesica, part or peptide can include but not limited to IF1 α, VEGF, PDGFRA or their any combination.

The present invention also provides the vesica separated, and it comprises one or more RCC specific biological mark, for example ALPHA-TFEB, NONO-TFE3, PRCC-TFE3, SFPQ-TFE3, CLTC-TFE3 or MALAT1-TFEB; Perhaps in Figure 51 and Fig. 1 for RCC listed those biomarkers.The composition of the vesica that comprises separation also is provided in addition.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more RCC specific biological mark, for example ALPHA-TFEB, NONO-TFE3, PRCC-TFE3, SFPQ-TFE3, CLTC-TFE3 or MALAT1-TFEB; Perhaps in Figure 51 and Fig. 1 for RCC listed biomarker.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony for example, is homogeneous basically for RCC specificity vesica or the vesica that comprises one or more RCC specific biological marks (in ALPHA-TFEB, NONO-TFE3, PRCC-TFE3, SFPQ-TFE3, CLTC-TFE3 or MALAT1-TFEB or Figure 51 and Fig. 1 for RCC listed biomarker).

For characterizing RCC, can also detect one or more RCC specific biological marks (for example ALPHA-TFEB, NONO-TFE3, PRCC-TFE3, SFPQ-TFE3, CLTC-TFE3 or MALAT1-TFEB by one or more systems disclosed herein; Perhaps in Figure 51 and Fig. 1 for RCC listed biomarker).For example, detection system can comprise that one or more probes for example, with one or more RCC specific biological marks of one or more vesicas in the detection of biological sample (ALPHA-TFEB, NONO-TFE3, PRCC-TFE3, SFPQ-TFE3, CLTC-TFE3 or MALAT1-TFEB; Perhaps in Figure 51 and Fig. 1 for RCC listed biomarker).

Liver cirrhosis

Liver cirrhosis specific biological mark from vesica (for example can comprise one or more, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) miR that cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Figure 52, and can be for setting up the liver cirrhosis specific biological marking.One or more mRNA that can analyze include but not limited to NLT, and it can be used as from the non-specific biomarker of the liver cirrhosis of vesica.

The protein that can be assessed in vesica, part or peptide can include but not limited to NLT, HBsAG, AST, YKL-40, hyaluronic acid, TIMP-1, alpha2 Macroglobulin, a-1-antitrypsin PlZ allelotrope, haptoglobin or acid phosphatase ACPAC or their any combination.

The present invention also provides the vesica separated, and it comprises one or more liver cirrhosis specific biological marks, for example in Figure 52 and Fig. 1 for liver cirrhosis listed biomarker.The composition of the vesica that comprises separation also is provided in addition.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more liver cirrhosis specific biological marks, for example in Figure 52 and Fig. 1 for liver cirrhosis listed biomarker.Described composition can comprise the vesica colony of obvious enrichment, wherein said vesica colony is for liver cirrhosis specificity vesica or comprise one or more liver cirrhosis specific biological marks, and for example in Figure 52 and Fig. 1, for liver cirrhosis, the vesica of listed biomarker is homogeneous basically.

For characterizing liver cirrhosis, can also detect one or more liver cirrhosis specific biological marks (for example in Figure 52 and Fig. 1 for liver cirrhosis listed biomarker) by one or more systems disclosed herein.For example, detection system can comprise that one or more probes for example, with one or more liver cirrhosis specific biological marks of one or more vesicas in the detection of biological sample (in Figure 52 and Fig. 1 for liver cirrhosis listed biomarker).

The esophageal carcinoma

Esophageal carcinoma specific biological mark from vesica (for example can comprise one or more, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) miR that cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Figure 53, and can be for setting up the esophageal carcinoma specific biological marking.For example, the described biological marking can comprise that one or more cross the miR of expression, such as but not limited to miR-192, miR-194, miR-21, miR-200c, miR-93, miR-342, miR-152, miR-93, miR-25, miR-424 or miR-151 or their any combination.The described biological marking can also comprise that one or more express not enough miR, such as but not limited to miR-27b, miR-205, miR-203, miR-342, let-7c, miR-125b, miR-100, miR-152, miR-192, miR-194, miR-27b, miR-205, miR-203, miR-200c, miR-99a, miR-29c, miR-140, miR-103 or miR-107 or their any combination.One or more mRNA that can analyze include but not limited to MTHFR, and can be as the specific biological mark of the esophageal carcinoma from vesica.

The present invention also provides the vesica separated, and it comprises one or more esophageal carcinoma specific biological marks, for example in Figure 53 and Fig. 1 for the esophageal carcinoma listed biomarker.The composition of the vesica that comprises separation also is provided in addition.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more esophageal carcinoma specific biological marks, for example in Figure 53 and Fig. 1 for the esophageal carcinoma listed biomarker.Described composition can comprise the vesica colony of obvious enrichment, wherein said vesica colony is for esophageal carcinoma specificity vesica or comprise one or more esophageal carcinoma specific biological marks, and for example in Figure 53 and Fig. 1, for the esophageal carcinoma, the vesica of listed biomarker is homogeneous basically.

For characterizing the esophageal carcinoma, can also detect one or more esophageal carcinoma specific biological marks (for example in Figure 53 and Fig. 1 for the esophageal carcinoma listed biomarker) by one or more systems disclosed herein.For example, detection system can comprise that one or more probes for example, with one or more esophageal carcinoma specific biological marks of one or more vesicas in the detection of biological sample (in Figure 53 and Fig. 1 for the esophageal carcinoma listed biomarker).

Cancer of the stomach

Cancer of the stomach specific biological mark from vesica (for example can comprise one or more, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) miR that cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Figure 54, and can be for setting up the cancer of the stomach specific biological marking.For example, the described biological marking can comprise that one or more cross the miR of expression, such as but not limited to miR-106a, miR-21, miR-191, miR-223, miR-24-1, miR-24-2, miR-107, miR-92-2, miR-214, miR-25 or miR-221 or their any combination.The described biological marking can also comprise that one or more express not enough miR, such as but not limited to let-7a.

One or more mRNA that can analyze include but not limited to RRM2, EphA4 or Survivin or their any combination, and can be as the cancer of the stomach specific biological mark from vesica.The biomarker sudden change of the cancer of the stomach that can be assessed in vesica includes but not limited to the sudden change of APC; Perhaps any combination of the specific sudden change of cancer of the stomach.The protein that can be assessed in vesica, part or peptide can include but not limited to EphA4.

The present invention also provides the vesica separated, and it comprises one or more cancer of the stomach specific biological marks, for example listed in Figure 54.The composition of the vesica that comprises separation also is provided in addition.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more cancer of the stomach specific biological marks, for example listed in Figure 54.Described composition can comprise the vesica colony of obvious enrichment, wherein said vesica colony is for cancer of the stomach specificity vesica or the vesica that comprises one or more cancer of the stomach specific biological marks, listed biomarker in Figure 54 for example, for homogeneous basically.

For characterizing cancer of the stomach, can also detect one or more cancer of the stomach specific biological marks (for example listed in Figure 54) by one or more systems disclosed herein.For example, detection system can comprise that one or more probes for example, with one or more cancer of the stomach specific biological marks of one or more vesicas in the detection of biological sample (in Figure 54 listed biomarker).

Autism

Autism specific biological mark from vesica (for example can comprise one or more, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) miR that cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Figure 55, and can be for setting up the autism specific biological marking.For example, the described biological marking can comprise that one or more cross the miR of expression, such as but not limited to miR-484, miR-21, miR-212, miR-23a, miR-598, miR-95, miR-129, miR-431, miR-7, miR-15a, miR-27a, miR-15b, miR-148b, miR-132 or miR-128 or their any combination.The described biological marking can also comprise that one or more express not enough miR, such as but not limited to miR-93, miR-106a, miR-539, miR-652, miR-550, miR-432, miR-193b, miR-181d, miR-146b, miR-140, miR-381, miR-320a or miR-106b or their any combination.The protein that can be assessed in vesica, part or peptide can include but not limited to GM1, GDla, GDlb or GTlb or their any combination.

The present invention also provides the vesica separated, and it comprises one or more autism specific biological marks, for example in Figure 55 and Fig. 1 for autism listed biomarker.The composition of the vesica that comprises separation also is provided in addition.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more autism specific biological marks, for example in Figure 55 and Fig. 1 for autism listed biomarker.Described composition can comprise the vesica colony of obvious enrichment, wherein said vesica colony is for autism specificity vesica or comprise one or more autism specific biological marks, and for example in Figure 55 and Fig. 1, for autism, the vesica of listed biomarker is homogeneous basically.

For characterizing autism, can also detect one or more autism specific biological marks (for example in Figure 55 and Fig. 1 for autism listed biomarker) by one or more systems disclosed herein.For example, detection system can comprise that one or more probes for example, with one or more autism specific biological marks of one or more vesicas in the detection of biological sample (in Figure 55 and Fig. 1 for autism listed biomarker).

Organ rejection response

Specific biological mark from the organ rejection response of vesica (for example can comprise one or more, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) miR that cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Figure 56, and can be for setting up the organ rejection response specific biological marking.For example, the described biological marking can comprise that one or more cross the miR of expression, such as but not limited to miR-658, miR-125a, miR-320, miR-381, miR-628, miR-602, miR-629 or miR-125a or their any combination.In addition, the described biological marking can also comprise that one or more express not enough miR, such as but not limited to miR-324-3p, miR-611, miR-654, miR-330MM1, miR-524, miR-17-3p MM1, miR-483, miR-663, miR-516-5p, miR-326, miR-197MM2 or miR-346 or their any combination.The protein that can be assessed in vesica, part or peptide can include but not limited to Matrix Metalloproteinase-9, protease 3 or HNP or their any combination.Described biomarker can be the member of matrix metalloproteinase.

The present invention also provides the vesica separated, and it comprises one or more organ rejection response specific biological marks, for example listed in Figure 56.The composition of the vesica that comprises separation also is provided in addition.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more organ rejection response specific biological marks, for example listed in Figure 56.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony for example, is homogeneous basically for organ rejection response specificity vesica or the vesica (listed in Figure 56) that comprises one or more organ rejection response specific biological marks.

For characterizing organ rejection response, can also detect one or more organ rejection response specific biological marks (for example listed in Figure 56) by one or more systems disclosed herein.For example, detection system can comprise one or more probes one or more organ rejection response specific biological marks (for example listed in Figure 56) with one or more vesicas in the detection of biological sample.

Methicillin-resistant staphylococcus aureus

Methicillin-resistant staphylococcus aureus specific biological mark from vesica (for example can comprise one or more, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) miR that cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Figure 57, and can be for setting up the methicillin-resistant staphylococcus aureus specific biological marking.

One or more mRNA that can analyze include but not limited to TSST-1, and it can be as the specific biological mark of the methicillin-resistant staphylococcus aureus from vesica.The biomarker sudden change of the methicillin-resistant staphylococcus aureus that can be assessed in vesica includes but not limited to the sudden change of mecA, a-protein SNP; Perhaps any combination of the specific mutant of methicillin-resistant staphylococcus aureus.The protein that can be assessed in vesica, part or peptide can include but not limited to ETA, ETB, TSST-1 or leueocidin or their any combination.

The present invention also provides the vesica separated, and it comprises one or more methicillin-resistant staphylococcus aureus specific biological marks, for example listed in Figure 57.The composition of the vesica that comprises separation also is provided in addition.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more methicillin-resistant staphylococcus aureus specific biological marks, for example listed in Figure 57.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony for example, is homogeneous basically for methicillin-resistant staphylococcus aureus specificity vesica or the vesica that comprises one or more methicillin-resistant staphylococcus aureus specific biological marks (listed in Figure 57).

For characterizing methicillin-resistant staphylococcus aureus, can also detect one or more methicillin-resistant staphylococcus aureus specific biological marks (for example listed in Figure 57) by one or more systems disclosed herein.For example, detection system can comprise one or more probes one or more methicillin-resistant staphylococcus aureus specific biological marks (for example listed in Figure 57) with one or more vesicas in the detection of biological sample.

Vulnerable plaque

Vulnerable plaque specific biological mark from vesica (for example can comprise one or more, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds or more kinds of) miR that cross to express, express not enough miR, mRNA, transgenation, protein, part, peptide, snoRNA or their any combination, for example listed in Figure 58, and can be for setting up the vulnerable plaque specific biological marking.The protein that can be assessed in vesica, part or peptide can include but not limited to IL-6, MMP-9, PAPP-A, DDi, Fibrinogen, Lp-PLA2, SCD40L, Il-18, oxLDL, GPx-1, MCP-1, PIGF or CRP or their any combination.

The invention provides the vesica of separation, it comprises one or more vulnerable plaque specific biological marks, for example in Figure 58 and Fig. 1 for vulnerable plaque listed biomarker.The composition of the vesica that comprises separation also is provided.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more vulnerable plaque specific biological marks, for example in Figure 58 and Fig. 1 for vulnerable plaque listed biomarker.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony for example, is homogeneous basically for vulnerable plaque specificity vesica or the vesica that comprises one or more vulnerable plaque specific biological marks (in Figure 58 and Fig. 1 for vulnerable plaque listed biomarker).

For characterizing vulnerable plaque, can also detect one or more vulnerable plaque specific biological marks (for example in Figure 58 and Fig. 1 for vulnerable plaque listed biomarker) by one or more systems disclosed herein.For example, detection system can comprise that one or more probes for example, with one or more vulnerable plaque specific biological marks of one or more vesicas in the detection of biological sample (in Figure 58 and Fig. 1 for vulnerable plaque listed biomarker).

Autoimmune disease

The present invention also provides the vesica separated, and it comprises one or more autoimmune disease specific biological marks, for example in Fig. 1 for autoimmune disease listed biomarker.The composition of the vesica that comprises separation also is provided.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more autoimmune disease specific biological marks, for example in Fig. 1 for autoimmune disease listed biomarker.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony for example, is homogeneous basically for autoimmune disease specificity vesica or the vesica that comprises one or more autoimmune disease specific biological marks (in Fig. 1 for autoimmune disease listed biomarker).

For characterizing autoimmune disease, can also detect one or more autoimmune disease specific biological marks (for example in Fig. 1 for autoimmune disease listed biomarker) by one or more systems disclosed herein.For example, detection system can comprise that one or more probes for example, with one or more autoimmune disease specific biological marks of one or more vesicas in the detection of biological sample (in Fig. 1 for autoimmune disease listed biomarker).

Tuberculosis (TB)

The present invention also provides the vesica separated, and it comprises one or more TB disease specific biomarkers, for example listed for the TB disease in Fig. 1.The composition of the vesica that comprises separation also is provided.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more TB disease specific biomarkers, for example in Fig. 1 for the TB disease listed biomarker.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony for example, is homogeneous in fact for TB disease specific vesica or the vesica that comprises one or more TB disease specific biomarkers (in Fig. 1 for the TB disease listed biomarker).

For characterizing the TB disease, can also detect one or more TB disease specific biomarkers (for example in Fig. 1 for the TB disease listed biomarker) by one or more systems disclosed herein.For example, detection system can comprise that one or more probes for example, with one or more TB disease specific biomarkers of one or more vesicas in the detection of biological sample (in Fig. 1 for the TB disease listed biomarker).

HIV

The present invention also provides the vesica separated, and it comprises one or more HIV disease specific biomarkers, for example in Fig. 1 for the HIV disease listed biomarker.The composition of the vesica that comprises separation also is provided.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more HIV disease specific biomarkers, for example in Fig. 1 for the HIV disease listed biomarker.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony for example, is homogeneous in fact for HIV disease specific vesica or the vesica that comprises one or more HIV disease specific biomarkers (in Fig. 1 for the HIV disease listed biomarker).

For characterizing the HIV disease, can also detect one or more HIV disease specific biomarkers (for example in Fig. 1 for the HIV disease listed biomarker) by one or more systems disclosed herein.For example, detection system can comprise that one or more probes for example, with one or more HIV disease specific biomarkers of one or more vesicas in the detection of biological sample (in Fig. 1 for the HIV disease listed biomarker).

Described one or more biomarkers can also be miRNA, for example raise or cross the miRNA expressed.The miRNA of described rise can be miR-29a, miR-29b, miR-149, miR-378 or miR-324-5p.One or more biomarkers can also be hidden for characterizing HIV-1, for example, by one or more miRNA of assessment.Described miRNA can be miR-28, miR-125b, miR-150, miR-223 and miR-382, and raises.

Asthma

The present invention also provides the vesica separated, and it comprises one or more asthma disease specific biological marks, for example in Fig. 1 for asthma disease listed biomarker.The composition of the vesica that comprises separation also is provided.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more asthma disease specific biological marks, for example in Fig. 1 for asthma disease listed biomarker.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony for example, is homogeneous basically for asthma disease specificity vesica or the vesica that comprises one or more asthma disease specific biological marks (in Fig. 1 for asthma disease listed biomarker).

For characterizing asthma disease, can also detect one or more asthma disease specific biological marks (for example in Fig. 1 for asthma disease listed biomarker) by one or more systems disclosed herein.For example, detection system can comprise that one or more probes for example, with one or more asthma disease specific biological marks of one or more vesicas in the detection of biological sample (in Fig. 1 for asthma disease listed biomarker).

Lupus

The present invention also provides the vesica separated, and it comprises one or more lupus disease specific biomarkers, for example in Fig. 1 for the lupus disease listed biomarker.The composition of the vesica that comprises separation also is provided.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more lupus disease specific biomarkers, for example in Fig. 1 for the lupus disease listed biomarker.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony for example, is homogeneous in fact for lupus disease specific vesica or the vesica that comprises one or more lupus disease specific biomarkers (in Fig. 1 for the lupus disease listed biomarker).

For characterizing the lupus disease, can also detect one or more lupus disease specific biomarkers (for example in Fig. 1 for the lupus disease listed biomarker) by one or more systems disclosed herein.For example, detection system can comprise that one or more probes for example, with one or more lupus disease specific biomarkers of one or more vesicas in the detection of biological sample (in Fig. 1 for the lupus disease listed biomarker).

Influenza

The present invention also provides the vesica separated, and it comprises one or more influenza disease specific biomarkers, for example in Fig. 1 for the influenza disease listed biomarker.The composition of the vesica that comprises separation also is provided.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more influenza disease specific biomarkers, for example in Fig. 1 for the influenza disease listed biomarker.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony for example, is homogeneous basically for influenza disease specific vesica or the vesica that comprises one or more influenza disease specific biomarkers (in Fig. 1 for the influenza disease listed biomarker).

For characterizing the influenza disease, can also detect one or more influenza disease specific biomarkers (for example in Fig. 1 for the influenza disease listed biomarker) by one or more systems disclosed herein.For example, detection system can comprise that one or more probes for example, with one or more influenza disease specific biomarkers of one or more vesicas in the detection of biological sample (in Fig. 1 for the influenza disease listed biomarker).

Thyroid carcinoma

The present invention also provides the vesica separated, it comprises one or more thyroid carcinoma specific biological marks, for example distinctive AKAP9-BRAF of papillary thyroid carcinoma, CCDC6-RET, ERC1-RETM, GOLGA5-RET, HOOK3-RET, HRH4-RET, KTN 1-RET, NCOA4-RET, PCM1-RET, PRKARA1A-RET, RFG-RET, RFG9-RET, Ria-RET, TGF-NTRK1, TPM3-NTRK1, TPM3-TPR, TPR-MET, TPR-NTRK1, TRIM24-RET, TRIM27-RET or TRIM33-RET; The perhaps distinctive PAX8-PPARy of folliculus shape thyroid carcinoma.The composition of the vesica that comprises separation also is provided.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more thyroid carcinoma specific biological marks, for example in Fig. 1 for thyroid carcinoma listed biomarker.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony for example, is homogeneous basically for thyroid carcinoma specificity vesica or the vesica that comprises one or more thyroid carcinoma specific biological marks (in Fig. 1 for thyroid carcinoma listed biomarker).

For characterizing thyroid carcinoma, can also detect one or more thyroid carcinoma specific biological marks (for example in Fig. 1 for thyroid carcinoma listed biomarker) by one or more systems disclosed herein.For example, detection system can comprise that one or more probes for example, with one or more thyroid carcinoma specific biological marks of one or more vesicas in the detection of biological sample (in Fig. 1 for thyroid carcinoma listed biomarker).

Gene fusion

One or more biomarkers of assessing of vesica can be genetic fusant, for example listed one or more in Figure 59.Fusion gene is the heterozygous genes by independently gene is set up side by side before by two.This can or form by trans-splicing by chromosome translocation or inversion, deletion.The fusion gene of gained can cause abnormal time and the space expression of gene, for example causes cell growth factor, angiogenesis factor, tumor promoter or contributes to the tumour conversion of cell and the unconventionality expression of the other factors that tumour generates.Described fusion gene can be carcinogenic due to following juxtaposition: 1) be adjacent to cell growth factor, tumor promoter or promote the strong promoter zone of a gene of the coding region of other gene that cancer forms, thereby cause the genetic expression improved; Perhaps 2) due to the fusion of two heterogeneic coding regions, the chimeric protein that causes chimeric gene and obtain thus having abnormal activity.

The example of fusion gene is BCR-ABL, the characteristic molecule abnormality in its chronic lymphocytic leukemia that is～90% (CML) and acute leukemia subgroup (Kurzrock etc., Annals of Internal Medicine 2003; 138 (10): 819-830).BCR-ABL is because the transposition between karyomit(e) 9 and 22 causes.Described transposition is combined the 3 ' zone of 5th ' district of BCR gene and ABL1, thereby obtain chimeric BCR-ABL1 gene, its coding has protein (Mittleman etc., the Nature Reviews Cancer 2007 of the tyrosine kinase activity of constitutive activity; 7 (4): 233-245).Abnormal tyrosine kinase activity has caused cell signalling, Growth of Cells and cell survival, apoptosis resistance and the cytokine dependent/non-dependent of imbalance, all these causes leukemic pathologic, physiologic (Kurzrock etc., Annals of Internal Medicine 2003; 138 (10): 819-830).

Another fusion gene is IGH-MYC, the symbolic characteristic of its burkitt's lymphoma that is～80% (Oncologist 2006 such as Ferry; 11 (4): 375-83).Causing the reason event of this result is the transposition between karyomit(e) 8 and 14, thereby makes the strong promoter of c-Myc oncogene and immunoglobulin heavy chain gene adjacent, causes c-myc to cross expression (Mittleman etc., Nature Reviews Cancer 2007; 7 (4): 233-245).It is the critical event that lymphoma generates that c-myc resets, and this is because it causes lasting vegetative state.It has widely the effect (Oncologist 2006 such as Ferry for the process by cell cycle, cytodifferentiation, apoptosis and cell adhesion; 11 (4): 375-83).

The multiple fusion gene taken place frequently has been included in Mittleman database (cgap.nci.nih.gov/Chromosomes/Mitelman), and can in vesica, be assessed, and can be for characterizing phenotype.Described genetic fusant can be for characterizing hematologic malignancies or epithelial tumor.For example can detect that TMPRSS2-ERG, TMPRSS2-ETV and SLC45A3-ELK4 merge and for characterizing prostate cancer; And can detect ETV6-NTRK3 and ODZ4-NRG1 for characterizing mammary cancer.

In addition, the assessment fusion gene existence or do not exist or expression level can for diagnose phenotype (for example cancer) and monitor therapy react to select the treatment.For example, the existence of BCR-ABL fusion gene is not only for for diagnosing the feature of CML, and is the target of the Novartis medicine imatinib mesylate (Gleevec) (it is receptor tyrosine kinase inhibitors) that is used for the treatment of CML.The treatment of imatinib causes Molecular responses (disappearance of BCR-ABL+ hemocyte), and the progresson free survival improved in BCR-ABL+CML patient (Kantarjian etc., Clinical Cancer Research 2007; 13 (4): 1089-1097).

For the existence of genetic fusant, do not exist or expression level is assessed vesica and can is by the existence for genetic fusant, not exist or expression level is assessed heterogeneous vesica colony.Perhaps, the vesica of assessing can be derived from specific cell type, cell source specificity vesica for example, as described above.The example of application that can assess to characterize the syzygy of phenotype comprises following:

mammary cancer

In order to characterize mammary cancer, can assess vesica for the specific syzygy of one or more mammary cancer (including but not limited to ETV6-NTRK3).Described vesica can be derived from breast cancer cell.

lung cancer

In order to characterize lung cancer, can assess vesica for the specific syzygy of one or more lung cancer (including but not limited to RLF-MYCL1, TGF-ALK or CD74-ROS1).Described vesica can be derived from lung carcinoma cell.

prostate cancer

In order to characterize prostate cancer, can assess vesica for the syzygy (including but not limited to ACSL3-ETV1, C15ORF21-ETV1, FLJ35294-ETV1, HERV-ETV1, TMPRSS2-ERG, TMPRSS2-ETV1/4/5, TMPRSS2-ETV4/5, SLC5A3-ERG, SLC5A3-ETV1, SLC5A3-ETV5 or KLK2-ETV4) of one or more prostatic cancer specifics.Described vesica can be derived from prostate cancer cell.

the cancer of the brain

In order to characterize the cancer of the brain, can assess vesica for the specific syzygy of one or more cancer of the brains (including but not limited to GOPC-ROS1).Described vesica can be derived from brain cancer cell.

head and neck cancer

In order to characterize head and neck cancer, can assess vesica for the specific syzygy of one or more head and neck cancers (including but not limited to CHCHD7-PLAG1, CTNNB1-PLAG1, FHIT-HMGA2, HMGA2-NFIB, LIFR-PLAG1 or TCEA1-PLAG1).Described vesica can be derived from head and/or neck cancer cells.

renal cell carcinoma (RCC)

In order to characterize RCC, can assess vesica for the specific syzygy of one or more RCC (including but not limited to ALPHA-TFEB, NONO-TFE3, PRCC-TFE3, SFPQ-TFE3, CLTC-TFE3 or MALAT1-TFEB).Described vesica can be derived from the RCC cell.

thyroid carcinoma

In order to characterize thyroid carcinoma, can for the specific syzygy of one or more thyroid carcinomas, (include but not limited to: the distinctive AKAP9-BRAF of papillary carcinoma Tiroidina, CCDC6-RET, ERC1-RETM, GOLGA5-RET, HOOK3-RET, HRH4-RET, KTN1-RET, NCOA4-RET, PCM1-RET, PRKARA1A-RET, RFG-RET, RFG9-RET, Ria-RET, TGF-NTRK1, TPM3-NTRK1, TPM3-TPR, TPR-MET, TPR-NTRK1, TRIM24-RET, TRIM27-RET or TRIM33-RET; The perhaps distinctive PAX8-PPARy of follicular thyroid carcinoma) assess vesica.Described vesica can be derived from thyroid carcinoma cell.

leukemia

In order to characterize leukemia, can for the specific amalgamation of one or more leukemia, (include but not limited to: the distinctive TTL-ETV6 of kemia (ALL), CDK6-MLL, CDK6-TLX3, ETV6-FLT3, ETV6-RUNX1, ETV6-TTL, MLL-AFF1, MLL-AFF3, MLL-AFF4, MLL-GAS7, TCBA1-ETV6, TCF3-PBX 1 or TCF3-TFPT, the distinctive BCL11B-TLX3 of T cell acute lymphoblastic leukemia (T-ALL), IL2-TNFRFS17, NUP214-ABL1, NUP98-CCDC28A, TAL1-STIL or ETV6-ABL2, the distinctive ATIC-ALK of Anaplastic large cell knurl (ALCL), KIAA1618-ALK, MSN-ALK, MYH9-ALK, NPM1-ALK, TGF-ALK or TPM3-ALK, the distinctive BCR-ABL1 of chronic lymphocytic leukemia (CML), BCR-JAK2, ETV6-EVI1, ETV6-MN1 or ETV6-TCBA1, the distinctive CBFB-MYH11 of AML, CHIC2-ETV6, ETV6-ABL1, ETV6-ABL2, ETV6-ARNT, ETV6-CDX2, ETV6-HLXB9, ETV6-PER1, MEF2D-DAZAP1, AML-AFF1, MLL-ARHGAP26, MLL-ARHGEF12, MLL-CASC5, MLL-CBL, MLL-CREBBP, MLL-DAB21P, MLL-ELL, MLL-EP300, MLL-EPS15, MLL-FNBP1, MLL-FOXO3A, MLL-GMPS, MLL-GPHN, MLL-MLLT1, MLL-MLLT11, MLL-MLLT3, MLL-MLLT6, MLL-MYO1F, MLL-PICALM, MLL-SEPT2, MLL-SEPT6, MLL-SORBS2, MYST3-SORBS2, MYST-CREBBP, NPM1-MLF1, NUP98-HOXA13, PRDM16-EVI1, RABEP1-PDGFRB, RUNX1-EVI1, RUNX1-MDS1, RUNX1-RPL22, RUNX1-RUNX1T1, RUNX1-SH3D19, RUNX1-USP42, RUNX1-YTHDF2, RUNX1-ZNF687 or TAF15-ZNF-384, the distinctive CCND1-FSTL3 of lymphocytic leukemia (CLL), the distinctive BCL3-MYC of B cell lymphocytic leukemia (B-CLL), MYC-BTG1, BCL7A-MYC, BRWD3-ARHGAP20 or BTG1-MYC, the distinctive CITTA-BCL6 of diffuse large B cell lymphoma (DLBCL), CLTC-ALK, IL21R-BCL6, PIM1-BCL6, TFCR-BCL6, IKZF1-BCL6 or SEC31A-ALK, eosinophilia/chronic eosinophilic increases distinctive FLIP1-PDGFRA, FLT3-ETV6, KIAA1509-PDGFRA, PDE4DIP-PDGFRB, NIN-PDGFRB, TP53BP1-PDGFRB or TPM3-PDGFRB, the distinctive IGH-MYC of Burkitt lymphoma or LCP1-BCL6) assess vesica.Described vesica can be derived from blood cell.

The present invention also provides the vesica separated, and it comprises one or more genetic fusants disclosed herein, for example listed in Figure 59.The composition of the vesica that comprises separation also is provided.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more genetic fusants, for example listed in Figure 59.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony for example, is homogeneous basically for the vesica that comprises one or more genetic fusants (listed in Figure 59).

This paper also provides the detection system for detection of one or more genetic fusants, for example listed genetic fusant in Figure 59.For example, detection system can comprise that one or more probes are to detect one or more listed genetic fusants in Figure 59.The detection of one or more genetic fusants can be for characterizing cancers.

Gene-correlation MiRNA biomarker

One or more biomarkers of assessing can also comprise one or more genes, and it is selected from PFKFB3, RHAMM (HMMR), cDNA FLJ42103, ASPM, CENPF, NCAPG, androgen receptor, EGFR, HSP90, SPARC, DNMT3B, GART, MGMT, SSTR3 and TOP2B.With the microRNA of one or more described gene interactions can be also biomarker (for example, referring to Figure 60).In addition, described one or more biomarkers can be for characterizing prostate cancer.

The present invention also provides the vesica separated, its comprise one or more biomarkers that formed by PFKFB3, RHAMM (HMMR), cDNA FLJ42103, ASPM, CENPF, NCAPG, androgen receptor, EGFR, HSP90, SPARC, DNMT3B, GART, MGMT, SSTR3 and TOP2B or with the microRNA (for example, referring to Figure 60) of these one or more gene interactions.The present invention further provides the composition of the vesica that comprises described separation.Therefore, in some embodiments, described composition comprises vesica colony, this colony comprises one or more biomarkers, and described biomarker is comprised of PFKFB3, RHAMM (HMMR), cDNA FLJ42103, ASPM, CENPF, NCAPG, androgen receptor, EGFR, HSP90, SPARC, DNMT3B, GART, MGMT, SSTR3 and TOP2B; Perhaps with the microRNA (for example, referring to Figure 60) of these one or more gene interactions.Described composition can comprise the vesica colony of obvious enrichment, wherein said vesica colony for comprise one or more biomarkers that formed by PFKFB3, RHAMM (HMMR), cDNA FLJ42103, ASPM, CENPF, NCAPG, androgen receptor, EGFR, HSP90, SPARC, DNMT3B, GART, MGMT, SSTR3 and TOP2B or with the vesica of the microRNA (for example, referring to Figure 60) of these one or more gene interactions be homogeneous basically.

Can also detect one or more prostatic cancer specific biomarkers (for example listed in Figure 60) by one or more systems disclosed herein.For example, detection system can comprise one or more probes one or more prostatic cancer specific biomarkers (for example listed in Figure 60) with one or more vesicas in the detection of biological sample.

With the interactional miRNA of PFKFB3 can be miR-513a-3p, miR-128, miR-488, miR-539, miR-658, miR-524-5p, miR-1258, miR-150, miR-216b, miR-377, miR-135a, miR-26a, miR-548a-5p, miR-26b, miR-520d-5p, miR-224, miR-1297, miR-1197, miR-182, miR-452, miR-509-3-5p, miR-548m, miR-625, miR-509-5p, miR-1266, miR-135b, miR-190b, miR-496, miR-616, miR-621, miR-650, miR-105, miR-19a, miR-346, miR-620, miR-637, miR-651, miR-1283, miR-590-3p, miR-942, miR-1185, miR-577, miR-602, miR-1305, miR-220c, miR-1270, miR-1282, miR-432, miR-491-5p, miR-548n, miR-765, miR-768-3p or miR-924, and can be used as biomarker.

The present invention also provides the vesica separated, and it comprises interactional one or more miRNA with PFKFB3.This paper also provides the composition of the vesica that comprises described separation.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more by the biomarker formed with the interactional miRNA of PFKFB3.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony is homogeneous basically for the vesica that comprises one or more and the interactional miRNA of PFKFB3.In addition, can also detect interactional one or more miRNA with PFKFB3 by one or more systems disclosed herein.For example, detection system can comprise one or more probes with one or more vesicas in the detection of biological sample with interactional one or more miRNA of PFKFB3.

With the interactional miRNA of RHAMM can be miR-936, miR-656, miR-105, miR-361-5p, miR-194, miR-374a, miR-590-3p, miR-186, miR-769-5p, miR-892a, miR-380, miR-875-3p, miR-208a, miR-208b, miR-586, miR-125a-3p, miR-630, miR-374b, miR-411, miR-629, miR-1286, miR-1185, miR-16, miR-200b, miR-671-5p, miR-95, miR-421, miR-496, miR-633, miR-1243, miR-127-5p, miR-143, miR-15b, miR-200c, miR-24 or miR-34c-3p.

The present invention also provides the vesica separated, and it comprises one or more and RHAMM interactional miRNA.The present invention further provides the composition of the vesica that comprises described separation.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more by the biomarker formed with the interactional miRNA of RHAMM.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony is homogeneous basically for the vesica that comprises one or more and the interactional miRNA of RHAMM.In addition, can also detect interactional one or more miRNA with RHAMM by one or more systems disclosed herein.For example, detection system can comprise one or more probes with one or more vesicas in the detection of biological sample with interactional one or more miRNA of RHAMM.

With the interactional miRNA of CENPF can be miR-30c, miR-30b, miR-190, miR-508-3p, miR-384, miR-512-5p, miR-548p, miR-297, miR-520f, miR-376a, miR-1184, miR-577, miR-708, miR-205, miR-376b, miR-520g, miR-520h, miR-519d, miR-596, miR-768-3p, miR-340, miR-620, miR-539, miR-567, miR-671-5p, miR-1183, miR-129-3p, miR-636, miR-106a, miR-1301, miR-17, miR-20a, miR-570, miR-656, miR-1263, miR-1324, miR-142-5p, miR-28-5p, miR-302b, miR-452, miR-520d-3p, miR-548o, miR-892b, miR-302d, miR-875-3p, miR-106b, miR-1266, miR-1323, miR-20b, miR-221, miR-520e, miR-664, miR-920, miR-922, miR-93, miR-1228, miR-1271, miR-30e, miR-483-3p, miR-509-3-5p, miR-515-3p, miR-519e, miR-520b, miR-520c-3p or miR-582-3p.

This paper also provides the vesica separated, and it comprises one or more and CENPF interactional miRNA.The present invention further provides the composition of the vesica that comprises described separation.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more by the biomarker formed with the interactional miRNA of CENPF.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony is homogeneous basically for the vesica that comprises one or more and the interactional miRNA of CENPF.In addition, can also detect interactional one or more miRNA with CENPF by one or more systems disclosed herein.For example, detection system can comprise one or more probes with one or more vesicas in the detection of biological sample with interactional one or more miRNA of CENPF.

With the interactional miRNA of NCAPG can be miR-876-5p, miR-1260, miR-1246, miR-548c-3p, miR-1224-3p, miR-619, miR-605, miR-490-5p, miR-186, miR-448, miR-129-5p, miR-188-3p, miR-516b, miR-342-3p, miR-1270, miR-548k, miR-654-3p, miR-1290, miR-656, miR-34b, miR-520g, miR-1231, miR-1289, miR-1229, miR-23a, miR-23b, miR-616 or miR-620.

The present invention also provides the vesica separated, and it comprises one or more and NCAPG interactional miRNA.The present invention further provides the composition of the vesica that comprises described separation.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more by the biomarker formed with the interactional miRNA of NCAPG.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony is homogeneous basically for the vesica that comprises one or more and the interactional miRNA of NCAPG.In addition, can also detect interactional one or more miRNA with NCAPG by one or more systems disclosed herein.For example, detection system can comprise one or more probes with one or more vesicas in the detection of biological sample with interactional one or more miRNA of NCAPG.

With the interactional miRNA of androgen receptor can be miR-124a, miR-130a, miR-130b, miR-143, miR-149, miR-194, miR-29b, miR-29c, miR-301, miR-30a-5p, miR-30d, miR-30e-5p, miR-337, miR-342, miR-368, miR-488, miR-493-5p, miR-506, miR-512-5p, miR-644, miR-768-5p or miR-801.

With the interactional miRNA of EGFR can be miR-105, miR-128a, miR-128b, miR-140, miR-141, miR-146a, miR-146b, miR-27a, miR-27b, miR-302a, miR-302d, miR-370, miR-548c, miR-574, miR-587 or miR-7.

The present invention also provides the vesica separated, and it comprises one or more and AR interactional miRNA.The present invention further provides the composition of the vesica that comprises described separation.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more by the biomarker formed with the interactional miRNA of AR.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony is homogeneous basically for the vesica that comprises one or more and the interactional miRNA of AR.In addition, can also detect interactional one or more miRNA with AR by one or more systems disclosed herein.For example, detection system can comprise one or more probes with one or more vesicas in the detection of biological sample with interactional one or more miRNA of AR.

With the interactional miRNA of HSP90 can be miR-1, miR-513a-3p, miR-548d-3p, miR-642, miR-206, miR-450b-3p, miR-152, miR-148a, miR-148b, miR-188-3p, miR-23a, miR-23b, miR-578, miR-653, miR-1206, miR-192, miR-215, miR-181b, miR-181d, miR-223, miR-613, miR-769-3p, miR-99a, miR-100, miR-454, miR-548n, miR-640, miR-99b, miR-150, miR-181a, miR-181c, miR-522, miR-624, miR-130a, miR-130b, miR-146, miR-148a, miR-148b, miR-152, miR-181a, miR-181b, miR-181c, miR-204, miR-206, miR-211, miR-212, miR-215, miR-223, miR-23a, miR-23b, miR-301, miR-31, miR-325, miR-363, miR-566, miR-9 or miR-99b.

The present invention also provides the vesica separated, and it comprises one or more and HSP90 interactional miRNA.The present invention further provides the composition of the vesica that comprises described separation.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more by the biomarker formed with the interactional miRNA of HSP90.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony is homogeneous basically for the vesica that comprises one or more and the interactional miRNA of HSP90.In addition, can also detect interactional one or more miRNA with HSP90 by one or more systems disclosed herein.For example, detection system can comprise one or more probes with one or more vesicas in the detection of biological sample with interactional one or more miRNA of HSP90.

With the interactional miRNA of SPARC can be miR-768-5p, miR-203, miR-196a, miR-569, miR-187, miR-641, miR-1275, miR-432, miR-622, miR-296-3p, miR-646, miR-196b, miR-499-5p, miR-590-5p, miR-495, miR-625, miR-1244, miR-512-5p, miR-1206, miR-1303, miR-186, miR-302d, miR-494, miR-562, miR-573, miR-10a, miR-203, miR-204, miR-211, miR-29, miR-29b, miR-29c, miR-339, miR-433, miR-452, miR-515-5p, miR-517a, miR-517b, miR-517c, miR-592 or miR-96.

The present invention also provides the vesica separated, and it comprises one or more and SPARC interactional miRNA.The present invention further provides the composition of the vesica that comprises described separation.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more by the biomarker formed with the interactional miRNA of SPARC.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony is homogeneous basically for the vesica that comprises one or more and the interactional miRNA of SPARC.In addition, can also detect interactional one or more miRNA with SPARC by one or more systems disclosed herein.For example, detection system can comprise one or more probes with one or more vesicas in the detection of biological sample with interactional one or more miRNA of SPARC.

With the interactional miRNA of DNMT3B can be miR-618, miR-1253, miR-765, miR-561, miR-330-5p, miR-326, miR-188, miR-203, miR-221, miR-222, miR-26a, miR-26b, miR-29a, miR-29b, miR-29c, miR-370, miR-379, miR-429, miR-519e, miR-598, miR-618 or miR-635.

The present invention also provides the vesica separated, and it comprises one or more and DNMT3B interactional miRNA.The present invention further provides the composition of the vesica that comprises described separation.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more by the biomarker formed with the interactional miRNA of DNMT3B.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony is homogeneous basically for the vesica that comprises one or more and the interactional miRNA of DNMT3B.In addition, can also detect interactional one or more miRNA with DNMT3B by one or more systems disclosed herein.For example, detection system can comprise one or more probes with one or more vesicas in the detection of biological sample with interactional one or more miRNA of DNMT3B.

With the interactional miRNA of GART can be miR-101, miR-141, miR-144, miR-182, miR-189, miR-199a, miR-199b, miR-200a, miR-200b, miR-202, miR-203, miR-223, miR-329, miR-383, miR-429, miR-433, miR-485-5p, miR-493-5p, miR-499, miR-519a, miR-519b, miR-519c, miR-569, miR-591, miR-607, miR-627, miR-635, miR-636 or miR-659.

The present invention also provides the vesica separated, and it comprises one or more and GART interactional miRNA.The present invention further provides the composition of the vesica that comprises described separation.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more by the biomarker formed with the interactional miRNA of GART.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony is homogeneous basically for the vesica that comprises one or more and the interactional miRNA of GART.In addition, can also detect interactional one or more miRNA with GART by one or more systems disclosed herein.For example, detection system can comprise one or more probes with one or more vesicas in the detection of biological sample with interactional one or more miRNA of GART.

With the interactional miRNA of MGMT can be miR-122a, miR-142-3p, miR-17-3p, miR-181a, miR-181b, miR-181c, miR-181d, miR-199b, miR-200a, miR-217, miR-302b, miR-32, miR-324-3p, miR-34a, miR-371, miR-425-5p, miR-496, miR-514, miR-515-3p, miR-516-3p, miR-574, miR-597, miR-603, miR-653, miR-655, miR-92, miR-92b or miR-99a.

The present invention also provides the vesica separated, and it comprises one or more and MGMT interactional miRNA.The present invention further provides the composition of the vesica that comprises described separation.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more by the biomarker formed with the interactional miRNA of MGMT.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony is homogeneous basically for the vesica that comprises one or more and the interactional miRNA of MGMT.In addition, can also detect interactional one or more miRNA with MGMT by one or more systems disclosed herein.For example, detection system can comprise one or more probes with one or more vesicas in the detection of biological sample with interactional one or more miRNA of MGMT.

With the interactional miRNA of SSTR3 can be miR-125a, miR-125b, miR-133a, miR-133b, miR-136, miR-150, miR-21, miR-380-5p, miR-504, miR-550, miR-671, miR-766 or miR-767-3p.

The present invention also provides the vesica separated, and it comprises one or more and SSTR3 interactional miRNA.The present invention further provides the composition of the vesica that comprises described separation.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more by the biomarker formed with the interactional miRNA of SSTR3.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony is homogeneous basically for the vesica that comprises one or more and the interactional miRNA of SSTR3.In addition, can also detect interactional one or more miRNA with SSTR3 by one or more systems disclosed herein.For example, detection system can comprise one or more probes with one or more vesicas in the detection of biological sample with interactional one or more miRNA of SSTR3.

With the interactional miRNA of TOP2B can be miR-548f, miR-548a-3p, miR-548g, miR-513a-3p, miR-548c-3p, miR-101, miR-653, miR-548d-3p, miR-575, miR-297, miR-576-3p, miR-548b-3p, miR-624, miR-548n, miR-758, miR-1253, miR-1324, miR-23b, miR-320a, miR-320b, miR-1183, miR-1244, miR-23a, miR-451, miR-568, miR-1276, miR-548e, miR-590-3p, miR-1, miR-101, miR-126, miR-129, miR-136, miR-140, miR-141, miR-144, miR-147, miR-149, miR-18, miR-181b, miR-181c, miR-182, miR-184, miR-186, miR-189, miR-191, miR-19a, miR-19b, miR-200a, miR-206, miR-210, miR-218, miR-223, miR-23a, miR-23b, miR-24, miR-27a, miR-302, miR-30a, miR-31, miR-320, miR-323, miR-362, miR-374, miR-383, miR-409-3p, miR-451, miR-489, miR-493-3p, miR-514, miR-542-3p, miR-544, miR-548a, miR-548b, miR-548c, miR-548d, miR-559, miR-568, miR-575, miR-579, miR-585, miR-591, miR-598, miR-613, miR-649, miR-651, miR-758, miR-768-3p or miR-9.

In addition, this paper also provides the vesica separated, and it comprises one or more and TOP2B interactional miRNA.The present invention further provides the composition of the vesica that comprises described separation.Therefore, in some embodiments, described composition comprises vesica colony, and this colony comprises one or more by the biomarker formed with the interactional miRNA of TOP2B.Described composition can comprise the vesica colony of obvious enrichment, and wherein said vesica colony is homogeneous basically for the vesica that comprises one or more and the interactional miRNA of TOP2B.In addition, can also detect interactional one or more miRNA with TOP2B by one or more systems disclosed herein.For example, detection system can comprise one or more probes with one or more vesicas in the detection of biological sample with interactional one or more miRNA of TOP2B.

Other microRNA biomarker

Can detect in vesica or assess and include but not limited to hsa-let-7a for other microRNA that characterizes phenotype, hsa-let-7b, hsa-let-7c, hsa-let-7d, hsa-let-7e, hsa-let-7f, hsa-miR-15a, hsa-miR-16, hsa-miR-17-5p, hsa-miR-17-3p, hsa-miR-18a, hsa-miR-19a, hsa-miR-19b, hsa-miR-20a, hsa-miR-21, hsa-miR-22, hsa-miR-23a, hsa-miR-189, hsa-miR-24, hsa-miR-25, hsa-miR-26a, hsa-miR-26b, hsa-miR-27a, hsa-miR-28, hsa-miR-29a, hsa-miR-30a-5p, hsa-miR-30a-3p, hsa-miR-31, hsa-miR-32, hsa-miR-33, hsa-miR-92, hsa-miR-93, hsa-miR-95, hsa-miR-96, hsa-miR-98, hsa-miR-99a, hsa-miR-100, hsa-miR-101, hsa-miR-29b, hsa-miR-103, hsa-miR-105, hsa-miR-106a, hsa-miR-107, hsa-miR-192, hsa-miR-196a, hsa-miR-197, hsa-miR-198, hsa-miR-199a, hsa-miR-199a ^*, hsa-miR-208, hsa-miR-129, hsa-miR-148a, hsa-miR-30c, hsa-miR-30d, hsa-miR-139, hsa-miR-147, hsa-miR-7, hsa-miR-10a, hsa-miR-10b, hsa-miR-34a, hsa-miR-181a, hsa-miR-181b, hsa-miR-181c, hsa-miR-182, hsa-miR-182 ^*, hsa-miR-183, hsa-miR-187, hsa-miR-199b, hsa-miR-203, hsa-miR-204, hsa-miR-205, hsa-miR-210, hsa-miR-211, hsa-miR-212, hsa-miR-181a ^*, hsa-miR-214, hsa-miR-215, hsa-miR-216, hsa-miR-217, hsa-miR-218, hsa-miR-219, hsa-miR-220, hsa-miR-221, hsa-miR-222, hsa-miR-223, hsa-miR-224, hsa-miR-200b, hsa-let-7g, hsa-let-7i, hsa-miR-1, hsa-miR-15b, hsa-miR-23b, hsa-miR-27b, hsa-miR-30b, hsa-miR-122a, hsa-miR-124a, hsa-miR-125b, hsa-miR-128a, hsa-miR-130a, hsa-miR-132, hsa-miR-133a, hsa-miR-135a, hsa-miR-137, hsa-miR-138, hsa-miR-140, hsa-miR-141, hsa-miR-142-5p, hsa-miR-142-3p, hsa-miR-143, hsa-miR-144, hsa-miR-145, hsa-miR-152, hsa-miR-153, hsa-miR-191, hsa-miR-9, hsa-miR-9 ^*, hsa-miR-125a, hsa-miR-126 ^*, hsa-miR-126, hsa-miR-127, hsa-miR-134, hsa-miR-136, hsa-miR-146a, hsa-miR-149, hsa-miR-150, hsa-miR-154, hsa-miR-154 ^*, hsa-miR-184, hsa-miR-185, hsa-miR-186, hsa-miR-188, hsa-miR-190, hsa-miR-193a, hsa-miR-194, hsa-miR-195, hsa-miR-206, hsa-miR-320, hsa-miR-200c, hsa-miR-155, hsa-miR-128b, hsa-miR-106b, hsa-miR-29c, hsa-miR-200a, hsa-miR-302a ^*, hsa-miR-302a, hsa-miR-34b, hsa-miR-34c, hsa-miR-299-3p, hsa-miR-301, hsa-miR-99b, hsa-miR-296, hsa-miR-130b, hsa-miR-30e-5p, hsa-miR-30e-3p, hsa-miR-361, hsa-miR-362, hsa-miR-363, hsa-miR-365, hsa-mir-302b ^*, hsa-miR-302b, hsa-miR-302c ^*, hsa-miR-302c, hsa-miR-302d, hsa-miR-367, hsa-miR-368, hsa-miR-369-3p, hsa-miR-370, hsa-miR-371, hsa-miR-372, hsa-miR-373 ^*, hsa-miR-373, hsa-miR-374, hsa-miR-375, hsa-miR-376a, hsa-miR-377, hsa-miR-378, hsa-miR-422b, hsa-miR-379, hsa-miR-380-5p, hsa-miR-380-3p, hsa-miR-381, hsa-miR-382, hsa-miR-383, hsa-miR-340, hsa-miR-330, hsa-miR-328, hsa-miR-342, hsa-miR-337, hsa-miR-323, hsa-miR-326, hsa-miR-151, hsa-miR-135b, hsa-miR-148b, hsa-miR-331, hsa-miR-324-5p, hsa-miR-324-3p, hsa-miR-338, hsa-miR-339, hsa-miR-335, hsa-miR-133b, hsa-miR-325, hsa-miR-345, hsa-miR-346, ebv-miR-BHRF1-1, ebv-miR-BHRF 1-2 ^*, ebv-miR-BHRF1-2, ebv-miR-BHRF1-3, ebv-miR-BART1-5p, ebv-miR-BART2, hsa-miR-384, hsa-miR-196b, hsa-miR-422a, hsa-miR-423, hsa-miR-424, hsa-miR-425-3p, hsa-miR-18b, hsa-miR-20b, hsa-miR-448, hsa-miR-429, hsa-miR-449, hsa-miR-450, hcmv-miR-UL22A, hcmv-miR-UL22A ^*, hcmv-miR-UL36, hcmv-miR-UL112, hcmv-miR-UL148D, hcmv-miR-US5-1, hcmv-miR-US5-2, hcmv-miR-US25-1, hcmv-miR-US25-2-5p, hcmv-miR-US25-2-3p, hcmv-miR-US33, hsa-miR-191 ^*, hsa-miR-200a ^*, hsa-miR-369-5p, hsa-miR-431, hsa-miR-433, hsa-miR-329, hsa-miR-453, hsa-miR-451, hsa-miR-452, hsa-miR-452 ^*, hsa-miR-409-5p, hsa-miR-409-3p, hsa-miR-412, hsa-miR-410, hsa-miR-376b, hsa-miR-483, hsa-miR-484, hsa-miR-485-5p, hsa-miR-485-3p, hsa-miR-486, hsa-miR-487a, kshv-miR-K12-10a, kshv-miR-K12-10b, kshv-miR-K12-11, kshv-miR-K12-1, kshv-miR-K12-2, kshv-miR-K12-9 ^*, kshv-miR-K12-9, kshv-miR-K12-8, kshv-miR-K12-7, kshv-miR-K12-6-5p, kshv-miR-K12-6-3p, kshv-miR-K12-5, kshv-miR-K12-4-5p, kshv-miR-K12-4-3p, kshv-miR-K12-3, kshv-miR-K12-3 ^*, hsa-miR-488, hsa-miR-489, hsa-miR-490, hsa-miR-491, hsa-miR-511, hsa-miR-146b, hsa-miR-202 ^*, hsa-miR-202, hsa-miR-492, hsa-miR-493-5p, hsa-miR-432, hsa-miR-432 ^*, hsa-miR-494, hsa-miR-495, hsa-miR-496, hsa-miR-193b, hsa-miR-497, hsa-miR-181d, hsa-miR-512-5p, hsa-miR-512-3p, hsa-miR-498, hsa-miR-520e, hsa-miR-515-5p, hsa-miR-515-3p, hsa-miR-519e ^*, hsa-miR-519e, hsa-miR-520f, hsa-miR-526c, hsa-miR-519c, hsa-miR-520a ^*, hsa-miR-520a, hsa-miR-526b, hsa-miR-526b ^*, hsa-miR-519b, hsa-miR-525, hsa-miR-525 ^*, hsa-miR-523, hsa-miR-518f ^*, hsa-miR-518f, hsa-miR-520b, hsa-miR-518b, hsa-miR-526a, hsa-miR-520c, hsa-miR-518c ^*, hsa-miR-518c, hsa-miR-524 ^*, hsa-miR-524, hsa-miR-517 ^*, hsa-miR-517a, hsa-miR-519d, hsa-miR-521, hsa-miR-520d ^*, hsa-miR-520d, hsa-miR-517b, hsa-miR-520g, hsa-miR-516-5p, hsa-miR-516-3p, hsa-miR-518e, hsa-miR-527, hsa-miR-518a, hsa-miR-518d, hsa-miR-517c, hsa-miR-520h, hsa-miR-522, hsa-miR-519a, hsa-miR-499, hsa-miR-500, hsa-miR-501, hsa-miR-502, hsa-miR-503, hsa-miR-504, hsa-miR-505, hsa-miR-513, hsa-miR-506, hsa-miR-507, hsa-miR-508, hsa-miR-509, hsa-miR-510, hsa-miR-514, hsa-miR-532, hsa-miR-299-5p, hsa-miR-18a ^*, hsa-miR-455, hsa-miR-493-3p, hsa-miR-539, hsa-miR-544, hsa-miR-545, hsa-miR-487b, hsa-miR-551a, hsa-miR-552, hsa-miR-553, hsa-miR-554, hsa-miR-92b, hsa-miR-555, hsa-miR-556, hsa-miR-557, hsa-miR-558, hsa-miR-559, hsa-miR-560, hsa-miR-561, hsa-miR-562, hsa-miR-563, hsa-miR-564, hsa-miR-565, hsa-miR-566, hsa-miR-567, hsa-miR-568, hsa-miR-551b, hsa-miR-569, hsa-miR-570, hsa-miR-571, hsa-miR-572, hsa-miR-573, hsa-miR-574, hsa-miR-575, hsa-miR-576, hsa-miR-577, hsa-miR-578, hsa-miR-579, hsa-miR-580, hsa-miR-581, hsa-miR-582, hsa-miR-583, hsa-miR-584, hsa-miR-585, hsa-miR-548a, hsa-miR-586, hsa-miR-587, hsa-miR-548b, hsa-miR-588, hsa-miR-589, hsa-miR-550, hsa-miR-590, hsa-miR-591, hsa-miR-592, hsa-miR-593, hsa-miR-595, hsa-miR-596, hsa-miR-597, hsa-miR-598, hsa-miR-599, hsa-miR-600, hsa-miR-601, hsa-miR-602, hsa-miR-603, hsa-miR-604, hsa-miR-605, hsa-miR-606, hsa-miR-607, hsa-miR-608, hsa-miR-609, hsa-miR-610, hsa-miR-611, hsa-miR-612, hsa-miR-613, hsa-miR-614, hsa-miR-615, hsa-miR-616, hsa-miR-548c, hsa-miR-617, hsa-miR-618, hsa-miR-619, hsa-miR-620, hsa-miR-621, hsa-miR-622, hsa-miR-623, hsa-miR-624, hsa-miR-625, hsa-miR-626, hsa-miR-627, hsa-miR-628, hsa-miR-629, hsa-miR-630, hsa-miR-631, hsa-miR-33b, hsa-miR-632, hsa-miR-633, hsa-miR-634, hsa-miR-635, hsa-miR-636, hsa-miR-637, hsa-miR-638, hsa-miR-639, hsa-miR-640, hsa-miR-641, hsa-miR-642, hsa-miR-643, hsa-miR-644, hsa-miR-645, hsa-miR-646, hsa-miR-647, hsa-miR-648, hsa-miR-649, hsa-miR-650, hsa-miR-651, hsa-miR-652, hsa-miR-548d, hsa-miR-661, hsa-miR-662, hsa-miR-663, hsa-miR-449b, hsa-miR-653, hsa-miR-411, hsa-miR-654, hsa-miR-655, hsa-miR-656, hsa-miR-549, hsa-miR-657, hsa-miR-658, hsa-miR-659, hsa-miR-660, hsa-miR-421, hsa-miR-542-5p, hcmv-miR-US4, hcmv-miR-UL70-5p, hcmv-miR-UL70-3p, hsa-miR-363 ^*, hsa-miR-376a ^*, hsa-miR-542-3p, ebv-miR-BART1-3p, hsa-miR-425-5p, ebv-miR-BART3-5p, ebv-miR-BART3-3p, ebv-miR-BART4, ebv-miR-BART5, ebv-miR-BART6-5p, ebv-miR-BART6-3p, ebv-miR-BART7, ebv-miR-BART8-5p, ebv-miR-BART8-3p, ebv-miR-BART9, ebv-miR-BART10, ebv-miR-BART11-5p, ebv-miR-BART11-3p, ebv-miR-BART12, ebv-miR-BART13, ebv-miR-BART14-5p, ebv-miR-BART14-3p, kshv-miR-K12-12, ebv-miR-BART15, ebv-miR-BART16, ebv-miR-BART17-5p, ebv-miR-BART17-3p, ebv-miR-BART18, ebv-miR-BART19, ebv-miR-BART20-5p, ebv-miR-BART20-3p, hsv1-miR-H1, hsa-miR-758, hsa-miR-671, hsa-miR-668, hsa-miR-767-5p, hsa-miR-767-3p, hsa-miR-454-5p, hsa-miR-454-3p, hsa-miR-769-5p, hsa-miR-769-3p, hsa-miR-766, hsa-miR-765, hsa-miR-768-5p, hsa-miR-768-3p, hsa-miR-770-5p, hsa-miR-802, hsa-miR-801 and hsa-miR-675.

For example, not bound by theory, miR-128A5, miR-129 and miR-128B are in the enrichment of brain camber; MiR-194, miR-148 and miR-192 are in the enrichment of liver camber; MIR-96, miR-150, miR-205, miR-182 and miR-183 are in the enrichment of thymus gland camber; MiR-204, miR-IOB5, miR-154 and miRl 34 are in the enrichment of testis camber; And miR-122, miR-210, miR-221, miR-141, miR-23A, miR-200C and miR-136 are in the enrichment of placenta camber.The biological marking that comprises one or more aforementioned miR can be used for distinguishing the patient's who suffers from cervical cancer, colorectal carcinoma and mammary cancer the positive and histopathological negative lymph nodes.

In another embodiment, the biological marking can comprise one or more in following miR: miR-125b-1, miR125b-2, miR-145, miR-21, miR-155, miR-10b, miR-009-1 (miR131-1), miR-34 (miR-170), miR-102 (miR-29b), miR-123 (miR-126), miR-140-as, miR-125a, miR-125b-1, miR-125b-2, miR-194, miR-204, miR-213, let-7a-2, let-7a-3, let-7d (let-7d-v1), let-7f-2, let-71 (let-7d-v2), miR-101-1, miR-122a, miR-128b, miR-136, miR-143, miR-149, miR-191, miR-196-1, miR-196-2, miR-202, miR-203, miR-206 and miR-210, it can be used for characterizing mammary cancer.

In another embodiment, detect in vesica that miR-375 expresses and for characterizing pancreas islet or acinar tumors.

In another embodiment again, can in vesica, detect one or more in following miR: miR-103-2, miR-107, miR-103-1, miR-342, miR-100, miR-24-2, miR-23a, miR-125a, miR-26a-1, miR-24-1, miR-191, miR-15a, miR-368, miR-26b, miR-125b-2, miR-125b-1, miR-26a-2, miR-335, miR-126.miR-1-2, miR-21, miR-25, miR-92-2, miR-130a, miR-93, miR-16-1, miR-145, miR-17, miR-99b, miR-181b-1, miR-146, miR-181b-2, miR-16-2, miR-99a, miR-197, miR-10a, miR-224, miR-92-1, miR-27a, miR-221, miR-320, miR-7-1, miR-29b-2, miR-150, miR-30d, miR-29a, miR-23b, miR-135a-2, miR-223, miR-3p21-v, miR-128b, miR-30b, miR-29b-1, miR-106b, miR-132, miR-214, miR-7-3, miR-29c, miR-367, miR-30c-2, miR-27b, miR-140, miR-10b, miR-20, miR-129-1, miR-340, miR-30a, miR-30c-1, miR-106a, miR-32, miR-95, miR-222, miR-30e, miR-129-2, miR-345, miR-143, miR-182, miR-1-1, miR-133a-1, miR-200c, miR-194-1, miR-210, miR-181c, miR-192, miR-220, miR-213, miR-323 and miR-375, the high expression level of wherein said one or more miR or mistake are expressed and be can be used for characterizing carcinoma of the pancreas.

Can in vesica, detect in following miR one or more expression and for characterizing megakaryocytopoiesis: miR-101, miR-126, miR-99a, miR-99-prec, miR-106, miR-339, miR-99b, miR-149, miR-33, miR-135 and miR-20.

It is believed that cell proliferation and miR-31, miR-92, miR-99a, miR-100, miR-125a, miR-129, miR-130a, miR-150, miR-187, miR-190, miR-191, miR-193, miR-204, miR-210, miR-211, miR-212, miR-213, miR-215, miR-216, miR-217, miR-218, miR-224, miR-292, miR-294, miR-320, miR-324, miR-325, miR-326, miR-330, miR-331, miR-338, miR-341, miR-369, miR-370, et-7a, Let-7b, Let-7c, Let-7d, Let-7g, miR-7, miR-9, miR-10a, miR-10b, miR-15a, miR-18, miR-19a, miR-17-3p, miR-20, miR-23b, miR-25, miR-26a, miR-26a, miR-30e-5p, miR-31, miR-32, miR-92, miR-93, miR-100, miR-125a, miR-125b, miR-126, miR-127, miR-128, miR-129, miR-130a, miR-135, miR-138, miR-139, miR-140, miR-141, miR-143, miR-145, miR-146, miR-150, miR-154, miR-155, miR-181a, miR-182, miR-186, miR-187, miR-188, miR-190, miR-191, miR-193, miR-194, miR-196, miR-197, miR-198, miR-199, miR-201, miR-204, miR-216, miR-218, miR-223, miR-293, miR-291-3p, miR-294, miR-295, miR-322, miR-333, miR-335, miR-338, miR-341, miR-350, miR-369, miR-373, the expression of miR-410 and miR-412 is associated.Detection to one or more above-mentioned miR can be used for characterizing cancers.

Detect in vesica and be disclosed in U.S. Patent No. 7,642 for other example of the miR of characterizing cancers, 348 (it has described relevant to prostate cancer 3, the evaluation of the nucleotide sequence of 765 kinds of uniquenesses) and U.S. Patent No. 7, in 592,441, it has described the microRNA relevant to liver cancer.

Other microRNA of usually expressing in entity cancer (such as colorectal carcinoma, lung cancer, mammary cancer, cancer of the stomach, prostate cancer and carcinoma of the pancreas) also can detect and for characterizing cancers in vesica.For example, can in vesica, detect in following miR one or more and for characterizing entity cancer: miR-21, miR-17-5p, miR-191, miR-29b-2, miR-223, miR-128b, miR-199a-1, miR-24-1, miR-24-2, miR-146, miR-155, miR-181b-1, miR-20a, miR-107, miR-32, miR-92-2, miR-214, miR-30c, miR-25, miR-221 and miR-106a.

Other example of the microRNA that can detect in vesica is disclosed in PCT publication number No.WO2006126040, WO2006033020, WO2005116250 and WO2005111211, US publication No.US20070042982 and US20080318210; And, in EP publication number No.EP1784501A2 and EP1751311A2, it is incorporated to this paper separately by reference.

Biomarker detects

According to disclosed herein, existence, level or concentration that can be by detecting microRNA, vesica or other biomarker is the detection of biological marking qualitatively or quantitatively.Can use multiple technologies known to those of skill in the art to detect these biological marking compositions.For example, can pass through microarray analysis, polymerase chain reaction (the PCR) (method that comprises PCR-based, real-time polymerase chain reaction (RT-PCR) for example, quantitative real-time polymerase chain reaction (Q-PCR/qPCR) etc.), hybridization with the allele-specific probe, enzyme mutant detects, ligase chain reaction (LCR), oligonucleotide linking parsing (OLA), the fluidic cell heteroduple analysis, the chemical chop of mispairing, mass spectrum, nucleic acid sequencing, single strand conformation polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE), temperature gradient gel elec-trophoresis (TGGE) (TGGE), restrictive fragment length polymerphism, the serial analysis of genetic expression (SAGE) or their combine detection biomarker.The biomarker that can increase before detection, such as nucleic acid.Also can pass through immunoassay, immunoblotting, immunoprecipitation, Enzyme Linked Immunoadsorbent Assay (ELISA, EIA), radioimmunoassay (RIA), flow cytometry or electron microscopy (EM) detection of biological mark.

According to as herein described, can use trapping agent and the detection agent detection of biological marking.Trapping agent can comprise antibody, fit or identification biomarker and can be used for catching other entity of described biomarker.Can comprise the circulating biological mark by captive biomarker, as, the protein in the solution of body fluid, nucleic acid, lipid or biological composite.Similarly, described trapping agent can be used for catching vesica.Detection agent can comprise other entity that antibody or identification biomarker and can be used for detect described biomarker vesica or identification vesica and can be used for detecting vesica.In some embodiments, described detection agent is labeled and marker is detected, and detects therefrom described biomarker or vesica.Described detection agent can be wedding agent, as antibody or fit.In other embodiments, described detection agent comprises small molecules, such as the membrane protein labelling agent.For example, referring to, be disclosed in the membrane protein labelling agent in the U.S. Patent Publication No. US 2005/0158708 of Alroy etc.In embodiments, describe according to the present invention and separate or catch vesica, and by one or more membrane protein labelling agent for detection of described vesica.In many cases, the antigen of being identified by described trapping agent and detection agent or other vesica are partly interchangeable.As a limiting examples, consider the vesica that there is in its surface the cell source specific antigens and there is cancer specific antigen on its surface.In one case, described vesica can be used for the antibody of described cell source specific antigens and be caught (for example passing through the capture antibody mooring in substrate), and described vesica is used the antibody for described cancer specific antigen to be detected (for example, by using the described detection antibody of fluorochrome label and detecting the fluorescent radiation that described dyestuff is launched) subsequently.In another case, described vesica can be used for the antibody of described cancer specific antigen and be caught (for example, by described capture antibody is anchored in substrate), and described vesica is used the antibody for described cell source specific antigens to be detected (for example, by using the described detection antibody of fluorochrome label and detecting the fluorescent radiation that described dyestuff is launched) subsequently.

In some embodiments, trapping agent and detection agent are identified same biomarker.Can according to circumstances use this scheme.In one embodiment, described biomarker is enough to detect the target vesica, for example, is enough to catch cell source specificity vesica.In other embodiments, described biomarker is multi-functional, as, there is cell source specificity and cancer specific characteristic.Described biomarker can be coordinated to use for other biomarker of catching and detecting with same.

A kind of method of detection of biological mark comprises as described above from the biological sample purifying or separates heterogeneous vesica colony and carry out sandwich assay (sandwich assay).Can use trapping agent to catch the vesica in described colony.Described trapping agent can be capture antibody, such as primary antibody.Described capture antibody can be incorporated into substrate, for example array, hole or particle.Can use detection agent (such as detecting antibody) to detect to catch or the vesica of combination.For example, described detection antibody can be for the antigen of described vesica.Directly mark and the detection of described detection antibody.Perhaps, but described detection agent indirect labelling and detection, such as the secondary antibody by being connected with the enzyme of described detection agent reaction.Can add detection reagent or detection substrate, and detection reaction, for example, described in PCT publication number No.WO2009092386.In the illustrative example of described wedding agent in conjunction with Rab-5b and the combination of described detection agent or detection CD63 or cFLIP, described trapping agent can be that anti-Rab 5b antibody and described detection agent can be anti-CD 63 or anti-cFLIP antibody therein.In some embodiments, described trapping agent is in conjunction with CD9, PSCA, TNFR, CD63, B7H3, MFG-E8, EpCam, Rab, CD81, STEAP, PCSA, PSMA or 5T4.For example, described trapping agent can be the antibody for CD9, PSCA, TNFR, CD63, B7H3, MFG-E8, EpCam, Rab, CD81, STEAP, PCSA, PSMA or 5T4.Described trapping agent can also be the antibody for MFG-E8, annexin V, tissue factor, DR3, STEAP, epha2, TMEM211, unc93A, A33, CD24, NGAL, EpCam, MUC17, TROP2 or TETS.Described detection agent can be combination or the reagent that detects CD63, CD9, CD81, B7H3 or EpCam, such as the detection antibody for CD63, CD9, CD81, B7H3 or EpCam.The various various combination tunables of trapping agent and/or detection agent are used.In one embodiment, described trapping agent comprises PCSA, PSMA, B7H3 and optionally comprises EpCam, and described detection agent comprises one or more four transmembrane proteins, such as CD9, CD63 and CD81.In another embodiment, described trapping agent comprises TMEM211 and CD24, and described detection agent comprises one or more four transmembrane proteins, such as CD9, CD63 and CD81.In another embodiment, described trapping agent comprises CD66 and EpCam, and described detection agent comprises one or more four transmembrane proteins, such as CD9, CD63 and CD81.These four transmembrane proteins and/or other the general vesica mark that improve quantity can improve described detection signal in some cases.Also can use sandwich method to detect protein or other circulating biological mark.The useful load that can collect described vesica of catching and wherein comprised for analysis, as mRNA, microRNA, DNA and soluble proteins.

In some embodiments, described trapping agent combination or target are in EpCam, B7H3 or CD24, and one or more biomarkers that detect on described vesica are CD9 and/or CD63.In one embodiment, described trapping agent combination or target are in EpCam, and one or more biomarkers that detect on described vesica are CD9, EpCam and/or CD81.Single trapping agent can be selected from CD9, PSCA, TNFR, CD63, B7H3, MFG-E8, EpCam, Rab, CD81, STEAP, PCSA, PSMA or 5T4.Described single trapping agent can also be the antibody for DR3, STEAP, epha2, TMEM211, unc93A, A33, CD24, NGAL, EpCam, MUC17, TROP2, MFG-E8, TF, annexin V or TETS.In some embodiments, described single trapping agent is selected from PCSA, PSMA, B7H3, CD81, CD9 and CD63.

In other embodiments, described trapping agent target is in PCSA, and one or more biomarkers that detect on the vesica of catching are B7H3 and/or PSMA.In other embodiments, described trapping agent target is in PSMA, and one or more biomarkers that detect on the vesica of catching are B7H3 and/or PCSA.In other embodiments, described trapping agent target is in B7H3, and one or more biomarkers that detect on the vesica of catching are PSMA and/or PCSA.In other embodiments again, described trapping agent target is in CD63, and one or more biomarkers that detect on the vesica of catching are CD81, CD83, CD9 and/or CD63.Different trapping agent disclosed herein and biomarker combinations can be used for characterizing phenotype, such as detection, diagnosis or prognosis disease, as cancer.In some embodiments, can use target in the detection of trapping agent and CD9 and the CD63 of EpCam; Use target in the detection of trapping agent and B7H3 and the PSMA of PCSA; Perhaps with the trapping agent of CD63 and the detection of CD81, analyze vesica, thereby characterize prostate cancer.In other embodiments, use target in the trapping agent of CD9 and the detection coupling of CD63, vesica to be used for characterizing colorectal carcinoma in the trapping agent of CD63 and detection or the target of CD63.The technician should understand, and the target of trapping agent and detection agent is used interchangeably.In illustrative example, considered that target is in the trapping agent of PCSA and target in the detection agent of B7H3 and PSMA.Because whole these marks can be used for detecting the vesica in PCa source, can be by described trapping agent target in B7H3 or PSMA and can identify PCSA by detection agent.For example, in some embodiments, described detection agent target is in PCSA, and comprises B7H3 and/or PSMA for one or more biomarkers of catching described vesica.In other embodiments, described trapping agent target is in PSMA, and comprises B7H3 and/or PCSA for one or more biomarkers of catching described vesica.In other embodiments, described detection agent target is in B7H3, and comprises PSMA and/or PCSA for one or more biomarkers of catching described vesica.In some embodiments, the invention provides the trapping agent and/or the detection agent that use for PSMA, B7H3 and/or PCSA and detect the method for prostate cancer cell in body fluid.Described body fluid can comprise blood, and it comprises serum or blood plasma.Described body fluid can comprise ejaculation liquid or sperm.In other embodiments, detect the method for prostate cancer and further use trapping agent and/or the detection agent for CD81, CD83, CD9 and/or CD63.Described method also provides the method that characterizes the GI imbalance, it comprises and uses one or more in DR3, STEAP, epha2, TMEM211, unc93A, A33, CD24, NGAL, EpCam, MUC17, TROP2 and TETS to catch vesica, and uses one or more general vesica antigens (such as CD81, CD63 and/or CD9) to detect the vesica of catching.Other reagent can improve test performance, as, improving test accuracy or AUC, it is by providing extra biological resolving power and/or testing noise by reduction and realize.

Comprise the use planar substrates for the technology of detection of biological mark of the present invention, such as array (as biochip or microarray), it has and is fixed in described suprabasil molecule and usings as the trapping agent of assisting the detection of the particular organisms marking.Described array can be used as the part of the test kit for analyzing one or more biomarkers or vesica and provides.The molecule that biomarker shown in mentioned above and Fig. 3-60 and the antigen shown in Fig. 1 are identified can be included in for detection of in the array with diagnose the illness (comprise symptom before disease).In some embodiments, array comprises custom arrays, and it comprises through selecting to identify specifically the biomolecules of target organism mark.Can improve custom arrays to detect the biomarker that improves statistic property, as to multiple predictors (as, logistic regression, identification analysis or regression tree model) in the extra biomolecules identified of the biological marking of cross validation error rate that produce to improve.In some embodiments, built custom arrays with the biology of study of disease, situation or symptom and the biological marking limited under physiological status has been carried out to somatotype.Can be selected according to statistical standard for the mark that is included in described custom arrays, for example in distinguishing phenotype or physiological status, be there is the statistical significance of desired level.In some embodiments, the standard significance of selecting p value=0.05 is to get rid of or to be included in the biomolecules in described microarray.Described p value can be proofreaied and correct for multiple comparisons.As illustrative example, from from suffer from or the experimenter's of the disease that do not take a disease sample the nucleic acid that extracts can hybridize with high-density micro-array, described microarray is in conjunction with thousands of kinds of gene orders.And the nucleic acid that has or do not have between the sample of the described disease of tool remarkable different levels can be chosen as biomarker and whether have described disease to distinguish sample.Can build custom arrays to detect selecteed biomarker.In some embodiments, custom arrays comprises the low density microarray, its refer to and there is low quantity (as, tens of or hundreds of, but not thousands of kinds) the array of addressable wedding agent.Can in substrate, form the low density array.In some embodiments, the low density array of customization is used the pcr amplification in plate well, as, TaqMan

gene Expression Assays (Life Technologies Corporation, Carlsbad, the Applied Biosystems of CA).

The addressable locations that planar array comprises biomolecules with array format usually (for example note (pad), address or microbit are put).The size of array should depend on formation and the end-use of described array.Can prepare and comprise the array of 2 kinds of different molecules to thousands of kinds of differing moleculars.Usually, described array comprises 2 kinds to 100,000 kinds of as many as or more kinds of molecule, and this depends on end-use and the manufacture method of described array.Comprise at least one biomolecules for microarray of the present invention, this Molecular Identification or catch the biomarker be present in the target organism marking, for example microRNA or form other biomolecules or the vesica of the described biological marking.In some arrays, a plurality of substrates with similar and different composition have been used.Therefore, planar array can comprise a plurality of less substrates.

The present invention can use eurypalynous array with the detection of biological mark, for example, and the biomarker relevant to the biological marking of purpose.Available array or microarray include but not limited to DNA microarray (such as cDNA microarray, oligonucleotide microarray and SNP microarray), microRNA array, protein microarray, Antibody microarray, micro-array tissue, cell microarray (also being called the transfection microarray), chemical compound microarray and carbohydrate array (sugared array).These arrays are more at large described above.In some embodiments, microarray comprises biochip, and it provides the high-density immobilization array of identification molecule (as antibody), wherein biomarker is incorporated into and connects monitoring (as passed through fluorescence) in the ranks.Fig. 2 A shows illustrative configuration, wherein for the capture antibody mooring of target vesica antigen on surface.Use subsequently the vesica of catching for the detection antibody test of identical or different target vesica antigen.Under available and ideal situation, can use the described capture antibody of fit replacement of mooring.Show the fluoroscopic examination agent.Can use similarly other detection agent, for example, zymetology is reacted, can be detected nano particle, radio-labeled etc.In other embodiments, array comprises and participates in combining and the form of capture protein with the detection of being undertaken by mass spectrum (MS) by biological chemistry or molecular interaction.Can and can analyze its useful load from the described vesica of surperficial wash-out, as microRNA.

The array or the microarray that can be used for one or more biomarkers of the detection of biological marking can be according to being described in U.S. Patent No. 6,329,209; 6,365,418; 6,406,921; Method preparation in 6,475,808 and 6,475,809 and U.S. Patent application series No.10/884,269, it is incorporated to this paper separately in full by reference.Can use the method that is described in these patents custom arrays for the preparation of the specific choosing group that detects biomarker as herein described.Commercially available microarray also can be used for implementing method of the present invention, it includes but not limited to (the Santa Clara from Affymetrix, CA), Illumina (San Diego, CA), Agilent (Santa Clara, CA), those microarraies of Exiqon (Denmark) or Invitrogen (Carlsbad, CA).Custom arrays and/or commercialization array comprise for detecting as described herein the array of protein, nucleic acid and other biomolecules and entity (as cell, vesica, virus).

In some embodiments, the molecule on array to be fixed in comprises protein or peptide.The protein of one or more types can be fixed from the teeth outwards.In specific embodiments, the sex change of protein minimized, make the activity change of protein minimize or make the minimized method of interaction and material between protein and its fixing surface to fix described protein.

Available array surface can be any required shape, form or size.The unrestricted example on surface comprises chip, continuous surface, curved surfaces, flexible surface, film, flat board, thin slice or pipe.Surface can have a square micron to about 500cm ²area.Area, length and the width on surface can change according to the demand of analysis to be performed.The factor of need considering can comprise (such as) the needing etc. of the needs of the simplification of operation, the restriction that forms surperficial material, detection system, depositing system (such as the array instrument).

In specific embodiment, hope be with physical means separate many groups or a plurality of arrays in conjunction with island or fixing biomolecules: this physical sepn contributes to different groups or array are exposed to different target solution.Therefore, in specific embodiment, in the microwell plate in the hole of array in thering is any amount.In such embodiments, the effect on the surface that forms array can be played in the end in described hole, or during array can form and then be placed on hole on other surface.In specific embodiment, for example wherein use in the situation on the surface that does not there is hole, can form in conjunction with island or can by molecule fixing from the teeth outwards with liner on, it has steric hole so that they are corresponding to described island or biomolecules can be placed on surface.This liner is preferably hydraulic seal.Liner can be placed from the teeth outwards any time in preparing the process of array, and if be no longer essential to the isolation of group or array, can remove.

In some embodiments, fixing molecule can be in conjunction with one or more biomarkers or the vesica that exist in the biological sample contacted with fixed member.In some embodiments, described fixed member has been modified the molecule existed in one or more vesicas that are contacted with described fixed member, or is modified by this molecule.Contact to generally comprise with described sample described sample is covered on described array.

Can detect in solution or be fixed on modification or the combination of the molecule on array by detection technique known in the art.The example of these technology comprises immunological technique, for example competitive binding analysis and sandwich assay; The fluoroscopic examination that device use such as cofocal scanner, confocal microscope or the system based on CCD and the technology such as fluorescence, fluorescence polarization (FP), FRET (fluorescence resonance energy transfer) (FRET), total internal reflection fluorescent (TIRF), fluorescence correlation spectroscopy (FCS) are carried out; Colorimetric/spectroscopic techniques; Surface plasma resonance (can measure the variation of the material mass of being adsorbed by surface by this technology); Use radioisotopic technology, comprise traditional isotropic substance combination and scintillation proximity assay (SPA); Mass spectrum, for example substance assistant laser desorpted/MALDI-MS (MALDI) and MALDI flight time (TOF) mass spectrum; Ellipsometry, it is for measuring the optical means of protein film thickness; QCM (Quartz Crystal Microbalance) (QCM), it is for measuring the extremely sensitive method that is adsorbed on lip-deep quality of materials; Scan-probe microscopy, for example atomic force microscopy method (AFM) and scanning forces microscopy (SEM); And detect such technology such as electrochemistry, resistance technique, acoustic technique, microwave and IR/Raman.Such as referring to Mere L etc., " Miniaturized FRET assays and microfluidics:key components for ultra-high-throughput screening, " Drug Discovery Today 4 (8): 363-369 (1999) and the document of quoting thereof; Lakowicz J R, Principles of Fluorescence Spectroscopy, the 2nd edition, Plenum Press (1999) or Jain KK:Integrative Omics, Pharmacoproteomics, and Human Body Fluids.In:Thongboonkerd V edits, Proteomics of Human Body Fluids:Principles, Methods and Applications.Volume 1:Totowa, N.J.:Humana Press, 2007, each document all is incorporated herein by reference in full.

Microarray technology can analyze with mass spectrum (MS) and other instrument combines.The interface of mass spectral:mass spectrographic electron spray(ES) can with microfluidic device in kapillary integrated.For example, a kind of commercially available system comprises eTag report thing, and it is the fluorescent marker with electrophoretic mobility of uniqueness and good definition; Each marker by the key that can cut with the coupling of biological or chemical probe.The different mobility addressing of each eTag report thing makes the mixture of these markers can pass through capillary electrophoresis deconvolution and quantitative rapidly.This system can be carried out the analysis of genetic expression, protein expression and protein function to same sample simultaneously, Jain KK:Integrative Omics, Pharmacoproteomics, and Human Body Fluids.In:Thongboonkerd V edits, Proteomics of Human Body Fluids:Principles, Methods and Applications. the 1st volume: Totowa, N.J.:Humana Press, 2007, it is incorporated herein by reference in full.

Biochip can comprise the component for microfluid or nano-fluid analysis.Microfluidic device can be used for separating or analyzing biomarker, such as measuring the biological marking.Microfluid system allow to for separating of, catch or detect vesica, detect microRNA, detect one or more processes and other process microminiaturization and compartmentation in circulating biological mark, the detection of biological marking.Aspect at least one of described system, described microfluidic device can be used one or more detection reagent, and this detection reagent can be for detection of one or more biomarkers.In one embodiment, described device detects the biomarker on vesica that separate or combination.Various probes, antibody, protein or other wedding agent can be used for detecting the biomarker in microfluid system.Described detection agent can be fixed in the different compartments of described microfluidic device or each passage by described device enters in hybridization or detection reaction.

Vesica in microfluidic device can be in described microfluidic device cracking its inclusion is detected, such as protein or nucleic acid, as DNA or RNA, such as miRNA or mRNA.Described nucleic acid can increase or direct-detection in described microfluidic device before detection.Therefore, microfluid system also can be used to the detection of various different markers is carried out to Multiple detection.In one embodiment, in described microfluidic device, catch vesica, the vesica of catching is cleaved, and measures the biological marking from the microRNA of described vesica useful load.The described biological marking can further comprise for catching the trapping agent of described vesica.

Novel nanofabrication technique opened the bio-sensing application (it depends on the processing of high-density, accurate array, for example the chip based on Nucleotide and be called in addition the protein array of heterogeneous nano-array) possibility.Nano-fluid allows the amount of fluid analysis thing in microchip further is reduced to that to receive premium on currency flat, and chip used herein is called nano chips.(such as referring to Unger M etc., Biotechniques 1999; 27 (5): 1008-14, Kartalov EP etc., Biotechniques 2006; 40 (1): 85-90, each document is incorporated herein by reference in full.) at present, commercially available nano chips provide simple single step analysis, for example total cholesterol, gross protein or glucose analysis, it can, by sample is combined with reagent, mix also monitoring reaction and move.Gel-free analytical procedure (Cutillas etc., Proteomics, 2005 of based on liquid chromatography (LC), with nanometer LC, separating; 5:101-112 and Cutillas etc., Mol Cell Proteomics 2005; 4:1038-1051, each document is incorporated herein by reference in full) can be combined with nano chips.

This paper further provides the device for fast detecting of the particular organisms marking contributed in the detection of biological sample.Described device can be integrated in the preparation of biological sample and polymerase chain reaction (PCR) on chip.Described device can contribute to the particular organisms marking of vesica in the detection of biological sample, and the example is as Pipper etc., Angewandte Chemie, 47 (21), that p.3900-3904 in (2008), describes provides, and it is incorporated herein by reference in full.Can use the biological marking of introducing vesica for micro-/ nano electro-chemical systems (MEMS/NEMS) sensor and the oral fluid of diagnostic use, as at Li etc., described in Adv Dent Res 18 (1): 3-5 (2005), it is incorporated herein by reference in full.

As substituting of planar array, use the analysis (for example the mensuration based on pearl, as described herein) of particulate to be combined with flow cytometry.Can use multiparametric analysis or other high-throughout detection analysis (reporter molecules that it has used the pearl coating with cognate ligand and has had the given activity consistent with the high sensitivity automatic technology).In the analytical system based on pearl, be fixed for the wedding agent of biomarker or vesica on the addressable microballoon, for example, such as trapping agent (capture antibody).For the various wedding agents of each single binding analysis can with dissimilar microballoon (that is, microballon) coupling, and analytical reaction occur on the surface of microballoon, for example Figure 63 B is described.For the wedding agent of vesica, it can be the capture antibody with the pearl coupling.Dyeing microballoon with discrete fluorescence intensity loads their suitable wedding agent or capture probes independently.The not pearl on the same group of carrying different wedding agents can be gathered together as required, thereby form customization pearl array.Then, the pearl array is hatched to be analyzed with described sample in single reaction vessel.Operable or through adapt to the example for microfluidic device of the present invention include but not limited to described herein those.

Can use reporting system based on fluorescence to detect described biomarker and fixing capture molecules or the product of wedding agent and form (for example, referring to Figure 63 A-B).Described biomarker can be with the direct mark of fluorophore, or is detected by the second fluorescently-labeled biomolecules of catching.Can in flow cytometer, measure the strength of signal derived from the biomarker of catching.Flow cytometer can be at first by it independent color coding identify each microballoon.For example, the different pearls discrete fluorescence intensity that can dye, make each pearl with varying strength have different wedding agents.Described pearl can be used at least 2 kinds of different markers or dyestuff to carry out mark or dyeing.In some embodiments, use at least 3,4,5,6,7,8,9 or 10 kind of different marker carry out mark to described pearl.In addition, there is marker or the dyestuff that can also there is different ratios and composition more than the pearl of a kind of marker or dyestuff.Described pearl can carry out external marking or dyeing, or can have inherent fluorescence or signal tracer.

The amount of the biomarker of catching on each single pearl can be measured by the second color fluorescence of the target-specific to combination.This allows to be obtained by single sample the multiple quantitative of multiple target in same experiment.Microtitre ELISA process that can the relative standard is sensitivity, reliability and accuracy relatively, or can be improved.The benefit of the system based on pearl is that catch biomolecules or wedding agent and the independent coupling of different microballoons of vesica provide multiplexing ability.For example, according to Figure 63 C, describe, 5 kinds of different to be detected (being detected by the antibody for antigen, CD63 for example, CD9, CD81, B7H3 and EpCam) combination of biomarker and 20 kinds of biomarkers (for this mark, catching vesica (using capture antibody, for example, for the antibody of CD9, PSCA, TNFR, CD63, B7H3, MFG-E8, EpCam, Rab, CD81, STEAP, PCSA, PSMA, 5T4 and/or CD24)) can obtain about 100 kinds of combinations to be detected.According to shown as " EpCam 2 * ", " CD632 * " in Figure 63 C, for the Multiple Antibodies of single target, can be used for the detection of probe to various epi-positions.In another example, multiple analysis comprises using for the wedding agent of CD24 and catches vesica and use the wedding agent for CD9, CD63 and/or CD81 to detect the vesica of catching.Can use detection agent (such as antibody) to detect the vesica of catching.According to described herein, described detection reagent is mark directly or indirectly.

Can at least 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,25,50,75 or 100 kind of different biomarker carry out multiplexing.For example, can carry out with the particle of multiple difference mark the analysis of heterogeneous vesica colony.The particle that at least 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,25,30,40,50,75 or 100 species diversity marks can be arranged.Described particle can external marking (for example using label), or their marks inherently.The particle of each difference mark can with the trapping agent coupling of vesica, wedding agent for example, thus catch vesica.The capable of choosing multiple trapping agent is to characterize the target phenotype, and it comprises the trapping agent for general vesica biomarker, cell source specific biological mark and disease biomarker.Can detect by multiple wedding agent one or more biomarkers of the vesica of catching subsequently.Can carry out direct mark with auxiliary detection to described wedding agent.Perhaps, can pass through the described wedding agent of the second reagent mark.For example, described wedding agent can be the antibody for the biomarker on described vesica.Described wedding agent is connected with vitamin H.The second pack contains the streptavidin be connected with the report thing, and can be added into to detect described biomarker.In some embodiments, can at least 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,25,50,75 or 100 kind of different biomarker analyze caught vesica.For example, multiple detection agent (that is, the detection of the multiple biomarker of the vesica of catching or vesica colony) can increase the signal of gained, the sensitivity allow improved, specificity or these two, and use the sample of small amount.For example, with using lesser amt detection mark (as the unique identification thing), compare, use the detection of carrying out over a kind of general vesica mark can improve described signal.For describing, use is compared and can be improved described signal with any one detection carried out in described four transmembrane proteins of independent use for the wedding agent detection vesica of the mark of two or three in CD9, CD63 and/or CD81.

Method based on immunoassay or sandwich assay also can be used for detecting the biomarker of vesica.An example comprises ELISA.Wedding agent or trapping agent can be combined with hole.For example, the antibody for vesica antigen can be connected with hole.Can detect the biomarker on the vesica of catching according to method described herein.Figure 63 A shows the explanatory view of sandwich-like immunoassay.Capture antibody can be for target vesica antigen, as vesica biomarker, cell source mark or disease marker.In the figure, use the fluorescent-labeled antibody for target vesica antigen to detect the vesica of catching.Can use multiple capture antibody, as, in the location separably on array or the different holes of immunoassay plate, use.Described detection antibody can be identical for described capture antibody antigen, or can be for other mark.Described capture antibody can be replaced by alternative wedding agent, such as the fit or lectin of mooring, and/or described detection antibody can be replaced similarly, for example, by detectable (as, mark) fit, lectin or other conjugated protein or entity replace.In one embodiment, for one or more trapping agents of general vesica biomarker, cell source mark and/or disease marker, can together with detection agent for general vesica biomarker (such as four transmembrane proteins, including but not limited to CD9, CD63 and one or more in CD81), use.

Figure 63 D shows the explanatory view that the method according to this invention is analyzed vesica.Trapping agent is for catching vesica, and detection agent is for detection of caught vesica, and describedly catches and detect the level of antibody or exist for characterizing phenotype.Trapping agent, detection agent and sign phenotype can be as herein described any one.For example, trapping agent comprises that mooring is in suprabasil antibody or fit, its identification target vesica antigen, and detection agent comprises for the traget antibody of target vesica antigen or fit, and the sign phenotype comprises diagnosis, prognosis or treatment diagnosis to disease.At Figure 63 D i) shown in schematic diagram in, use and to catch vesica colony (6300) for one or more trapping agents of general vesica biomarker.Use subsequently for the detection agent (6301) of cell source biomarker and/or the vesica of catching for detection agent (6302) mark of disease specific biomarker.If only use cell source detection agent (6301), for the biological marking that characterizes described phenotype (6303), can comprise described general vesica mark (6300) and described cell source biomarker (6301).If only use disease detection agent (6302), for the biological marking that characterizes described phenotype (6303), can comprise described general vesica mark (6300) and described disease biomarker (6302).Perhaps, use detection agent to detect cell source biomarker (6301) and disease specific biomarker (6302).In this case, can comprise described general vesica mark (6300), described cell source biomarker (6301) and described disease biomarker (6302) for the biological marking that characterizes described phenotype (6403).Described biomarker combinations is through selecting to characterize the target phenotype and can be selected from biomarker as herein described and phenotype.

At Figure 63 D ii) shown in schematic diagram in, use and to catch vesica colony for one or more trapping agents of cell source biomarker (6310) and/or disease biomarker (6311).Use subsequently the detection agent (6312) for general vesica biomarker to detect the vesica of catching.If only used cell source trapping agent (6310), for the biological marking (6313) that characterizes described phenotype, can comprise described cell source biomarker (6310) and described general vesica mark (6312).If only used disease biomarker trapping agent (6311), for the biological marking (6313) that characterizes described phenotype, can comprise described disease biomarker (6311) and described general vesica biomarker (6312).Perhaps, the trapping agent for one or more cell source biomarkers (6310) and one or more disease specific biomarkers (6311) is used to catch vesica.In this case, can comprise described cell source biomarker (6310), described disease biomarker (6311) and described general vesica mark (6313) for the biological marking (6313) that characterizes described phenotype.Described biomarker combinations is through selecting to characterize the target phenotype and can be selected from biomarker as herein described and phenotype.

But analysis package contains the vesica useful load of biomarker to characterize phenotype.Useful load comprises biological entities, and it is included in the vesica film.These entities include but not limited to nucleic acid (as mRNA, microRNA or DNA fragmentation); Protein (as soluble protein and embrane-associated protein); Carbohydrate; Lipid; Metabolite; And various small molecules, as hormone.Described useful load can be the part of cellular environment, and it is encapsulated when vesica is formed at described cellular environment.In some embodiments of the present invention, also analyzed described useful load except detecting the vesica surface antigen.Can be according to the specific vesica colony that catches mentioned above, the useful load in the vesica caught subsequently can be used for characterizing phenotype.For example, can further be separated in the vesica of catching in the substrate useful load wherein with assessment.Perhaps, the vesica in sample is carried out detection and sorting and do not catch.Can further separate the useful load wherein with assessment of the vesica that detects by this mode.In one embodiment, by flow cytometry sorting vesica colony and analyzed the useful load in the vesica of institute's sorting.At Figure 63 E iii) shown in schematic diagram in, use one or more cell source biomarkers (6320), disease biomarker (6321) and general vesica mark (6322) to catch and/or detect vesica colony (6320).Assessed the useful load (6323) of the vesica separated.The biological marking detected in described useful load can be used for characterizing phenotype (6324).In a limiting examples, can use for the antibody of one or more target vesica antigens and analyze vesica colony in the plasma sample from the patient.Described antibody can be mooring in substrate to separate the capture antibody of required vesica colony.Perhaps, the direct mark and the vesica of institute's mark being separated by using flow cytometry to carry out sorting of described antibody.The microRNA extracted from described separated vesica colony or existence or the level of mRNA can be used for the detection of biological marking.The described biological marking is subsequently for diagnosis, prognosis or the described patient for the treatment of diagnosis.

In other embodiments, analyze the vesica useful load in the situation that at first do not catch or detect the vesica subgroup in vesica colony.For example, according to described herein, usually can use centrifugal, filtration, chromatogram or other technology to separate vesica from sample.After this can analyze the useful load of separated vesica with the detection of biological marking and sign phenotype.At Figure 63 E iv) shown in schematic diagram in, separated vesica colony (6330) and assessed the useful load (6331) of the vesica separated.The biological marking detected in described useful load can be used for characterizing phenotype (6332).In limiting examples, use size exclusion to separate vesica colony with membrane filtration from the plasma sample from the patient.The microRNA extracted from described vesica colony or the existence of mRNA or level are for detection of the biological marking.The described biological marking is subsequently for diagnosis, prognosis or the described patient for the treatment of diagnosis.

Can analyze peptide or protein biomarker by mass spectrum or flow cytometry.Can carry out according to program well-known in the art the Proteomic analysis of vesica by immunocytochemical stain, Western trace, electrophoresis, SDS-PAGE, chromatogram, x radiocrystallography or other oroteins analytical technology.In other embodiments, can use Chromy etc., J Proteome Res, 2004; 2D difference gel electrophoresis described in 3:1120-1127 (it is incorporated herein by reference in full) or use Zhang etc., Mol Cell Proteomics, 2005; Liquid chromatography mass described in 4:144-155 (it is incorporated herein by reference in full) is analyzed the biological marking of protein of vesica.Vesica can carry out the protein somatotype based on active, and it is described in, for example, and Berger etc., Am J Rharmacogenomics, 2004; In 4:371-381, it is incorporated herein by reference in full.In other embodiments, vesica can be used Pisitkun etc., Proc Natl Acad Sci U S A, 2004; Nano-spray liquid chromatography-tandem mass spectrometry described in 101:13368-13373 carrys out somatotype, and it is incorporated herein by reference in full.In another embodiment, vesica use tandem mass spectrum (MS) (liquid chromatography/MS/MS (LC-MS/MS) somatotype for example, its use, for example, LTQ and LTQ-FT ion trap mass spectrometer.Can be according to Smalley etc., J Proteome Res, 2008; The described relatively spectrum of 7:2088-2096 is counted and is determined the identity of protein and assess relative amount, and it is incorporated herein by reference in full.

Can also identify the expression of circulating protein matter biomarker or the protein useful load in vesica.Rear one after separating of analyzing optionally the specific vesica carried out at the trapping agent used for target acquistion colony carries out.In one embodiment, use immunocytochemical stain to express with analysing protein.Described sample can be resuspended in damping fluid, uses cytocentrifuge centrifugal under 100 * g (for example) 3 minutes on the viscosity slide, to prepare for immunocytochemical stain.Can be by cell centrifugation smear air-dry overnight, and be stored under-80 ℃ until dyeed.Then use the serum-free closed reagent to fix and seal slide.Afterwards, described slide is hatched together with specific antibodies, thus the expression of detection target protein.In some embodiments, described vesica not purified before carrying out protein expressioning analysis, separate or concentrated.

The vesica useful load that comprises the biological marking can be identified by metabolite mark or the metabolite analyzed in described vesica.Described the method for various metabolite orientations, for example the metabolite target is analyzed, metabolite somatotype or metabolite fingerprint, such as referring to Denkert etc., and Molecular Cancer 2008; 7:4598-4617, Ellis etc., Analyst 2006; 8:875-885, Kuhn etc., Clinical Cancer Research 2007; 24:7401-7406, Fiehn O., Comp Funct Genomics 2001; 2:155-168, Fancy etc., Rapid Commun Mass Spectrom 20 (15): 2271-80 (2006), Lindon etc., Pharm Res, 23 (6): 1075-88 (2006), Holmes etc., Anal Chem.2007 April 1; 79 (7): 2629-40.2007 electronic publishing on February 27, Erratum in:Anal Chem.2008 August 1; 80 (15): 6142-3, Stanley etc., Anal Biochem.2005 August 15; 343 (2): 195-202.,

deng, J Biol Chem.2003 November 14; 278 (46): 45915-23, each document is incorporated herein by reference in full.

Can pass through Jain KK:Integrative Omics, Pharmacoproteomics, and Human Body Fluids.In:Thongboonkerd V edits, Proteomics of Human Body Fluids:Principles, Methods and Applications.Volume 1:Totowa, N.J.:Humana Press, the system described in 2007 is analyzed peptide, and it is incorporated herein by reference in full.This system can be created on the sensitive molecular fingerprint of the protein existed in body fluid and vesica.Commercial applications (comprising the use in the benchmark library of stable metabolites all in Chromatography/Mass Spectrometry and human body, for example Paradigm Genetic ' s Human Metabolome Project) can be for determining the biological marking of metabolite.Can be included in U.S. Patent No. 6 for other method of analyzing the metabolism spectrum, 683, method and apparatus described in 455 (Metabometrix), U.S. Patent Application Publication No. No.20070003965 and 20070004044 (Biocrates Life Science), each document is incorporated herein by reference in full.Other oroteins component type technology is at Kennedy, Toxicol Lett 120:379-384 (2001), Berven etc., Curr Pharm Biotechnol 7 (3): 147-58 (2006), Conrads etc., Expert Rev Proteomics 2 (5): 693-703, Decramer etc., World J Urol 25 (5): 457-65 (2007), Decramer etc., Mol Cell Proteomics 7 (10): 1850-62 (2008), Decramer etc., Contrib Nephrol, 160:127-41 (2008), Diamandis, J Proteome Res 5 (9): 2079-82 (2006), Immler etc., Proteomics 6 (10): 2947-58 (2006), Khan etc., J Proteome Res 5 (10): 2824-38 (2006), Kumar etc., Biomarkers 11 (5): 385-405 (2006), Noble etc., Breast Cancer Res Treat 104 (2): 191-6 (2007), Omenn, Dis Markers 20 (3): 131-4 (2004), Powell etc., Expert Rev Proteomics 3 (1): 63-74 (2006), Rai etc., Arch Pathol Lab Med, 126 (12): 1518-26 (2002), Ramstrom etc., Proteomics, 3 (2): 184-90 (2003), Tammen etc., Breast Cancer Res Treat, 79 (1): 83-93 (2003), Theodorescu etc., Lancet Oncol, 7 (3): 230-40 (2006) or Zurbig etc., Electrophoresis, 27 (11): in 2111-25 (2006), describe to some extent.

For the analysis of mRNA, miRNA or other little RNA, can use for separating of any other known method of nucleic acid and separate total RNA, for example, in the method described in U.S. Patent Application Publication No. No.2008132694, it is incorporated herein by reference in full.Described method includes but not limited to that this test kit is commercially available for implementing the test kit of the RNA purifying based on film.Usually, test kit can be for being carried out small-scale RNA preparation (30mg or still less) by cell and tissue, for by cell and tissue, carrying out medium-scale RNA preparation (250mg tissue), and for carried out extensive RNA preparation (1g at most) by cell and tissue.Other commercially available test kit for total RNA that separation comprises little RNA effectively is also available.These methods can be used for isolating nucleic acid from vesica.

Perhaps, can use U.S. Patent No. 7,267, the method described in 950 is carried out isolation of RNA, and it is incorporated herein by reference in full.U.S. Patent No. 7,267,950 have described the method for extracting RNA in biosystem (cell, cell fragment, organoid, tissue, organ or organism), the solution that wherein will comprise RNA contacts with the combinable substrate of RNA, and by applying negative pressure by described substrate, reclaiming RNA.Perhaps, can use in the method described in U.S. Patent application No.20050059024 (it has described the separation of small RNA molecular) and carry out isolation of RNA, it is incorporated herein by reference in full.Other method is described to some extent in U.S. Patent application No.20050208510,20050277121,20070238118, and it all is incorporated to this paper with way of reference separately in full.

In one embodiment, can on the mRNA of the vesica by sample separation, carry out the mrna expression analysis.In some embodiments, described vesica is the specific vesica of cell source.The expression pattern produced by vesica can be indicated given morbid state, disease stage, the treatment dependency marking or physiological situation.

In one embodiment, once separate, obtain total RNA, can synthesize cDNA, and to carry out qRT-PCR for specific mRNA target according to the scheme of manufacturers and analyze (Applied Biosystem ' s Taqman for example analyze), or expressed the microarray expression mark collection multiplexing with height of observation in an experiment.Comprise the amount of measuring the RNA produced by gene (its can coded protein or peptide) for the method for setting up gene expression profile.This can complete by quantitative reverse transcriptase PCR (qRT-PCR), competitive RT-PCR, real-time RT-PCR, differential RT-PCR, Northern engram analysis or other dependence test.Although can react to implement described these technology with independent PCR, complementary DNA (cDNA) or the complementary RNA (cRNA) that can also increase and be produced by mRNA, and by microarray, it is analyzed.

Can use any suitable technology (including but not limited to the analysis of the Northern marking, RT-PCR, qRT-PCR, in situ hybridization or microarray analysis) that is applicable to mrna expression level in the detection of biological sample to carry out the level of miRNA product in measure sample.For example, by using primer and the target cDNA of gene specific, qRT-PCR has realized that the sensitive and quantitative miRNA that a small amount of target miRNA is carried out measures (by substance or multiple analysis), or can make platform be adapted to use 96 holes or 384 hole flat type to implement high-throughout measurement.Such as referring to Ross JS etc., Oncologist.2008 May; 13 (5): 477-93, it is incorporated herein by reference in full.A large amount of different array configurations and be known to those skilled in the art for generation of the method for microarray, and be described in United States Patent (USP), such as: U.S. Patent No. 5,445,934; 5,532,128; 5,556,752; 5,242,974; 5,384,261; 5,405,783; 5,412,087; 5,424,186; 5,429,807; 5,436,327; 5,472,672; 5,527,681; 5,529,756; 5,545,531; 5,554,501; 5,561,071; 5,571,639; 5,593,839; 5,599,695; 5,624,711; 5,658,734 or 5,700,637, it is incorporated to this paper in full with way of reference separately.Other method of miRNA somatotype is at Taylor etc., Gynecol Oncol.2008 July; 110 (1): 13-21, Gilad etc., PLoS ONE.2008 September 5; 3 (9): e3148, Lee etc., Annu Rev Pathol.2008 September 25 and Mitchell etc., Proc Natl Acad Sci U S is the moon 29 A.20087; 105 (30): 10513-8, Shen R etc., BMC Genomics.2004 December 14; 5 (1): 94, Mina L etc., Breast Cancer Res Treat.2007 June; 103 (2): 197-208, Zhang L etc., Proc Natl Acad Sci U S is on May 13, A.2008; 105 (19): 7004-9, Ross JS etc., Oncologist.2008 May; 13 (5): 477-93, Schetter AJ etc., JAMA.2008 January 30; 299 (4): 425-36, Staudt LM, N Engl J Med 2003; 348:1777-85, Mulligan G etc., Blood.2007 April 15; 109 (8): 3177-88.2006 electronic publishing on December 21, McLendon R etc., Nature.2008 October 23; 455 (7216): 1061-8, and describe to some extent in U.S. Patent No. 5,538,848,5,723,591,5,876,930,6,030,787,6,258,569 and 5,804,375, each document is incorporated herein by reference in full.In some embodiments, the microRNA group pattern is for synchronously inquiring after the expression of multiple miR.Exiqon mIRCURY LNA microRNA PCR system group (Exiqon, Inc., Wobum, MA) or from the TaqMan of Applied Biosystem

microRNA is analyzed and analytical system (Foster City, CA) can be used for these purposes.

Microarray technology allows to measure steady-state mRNA or the miRNA level of thousands of transcripts or miRNA simultaneously, and for example, powerful for the identification of effect outbreak, blocking-up or the regulation and control of uncontrolled cell proliferation (to) is provided thus.Can use the two microarray technologies such as cDNA array and oligonucleotide arrays.The result of these analyses is generally measuring of the strength of signal that received by label probe, and wherein said label probe is for detection of the cDNA sequence from sample, the nucleic acid array hybridizing of known location on this sequence and described microarray.The intensity of described signal amount common and cDNA is proportional, therefore proportional with the amount of the mRNA expressed in described sample cell or miRNA.These type of a large amount of technology can be with available.Be found in the U.S. Patent No. 6 of Linsley etc. for the method for measuring genetic expression, 271,002, the U.S. Patent No. 6 of Friend etc., 218,122, the U.S. Patent No. 6,218 of Peck etc., 114 or the U.S. Patent No. 6 of Wang etc., 004,755, it is incorporated herein by reference separately in full.

Can carry out the analysis of expression level by contrasting these intensity.This can be by generating gene in specimen expression intensity and control sample in the ratio matrix of expression intensity of gene carry out.Described control sample can be used as reference, and can use and consider age, other different references of race and sex.Different references can be for the different steps of different situations or disease and disease or situation and for determining curative effect.

For example, the genetic expression intensity that is derived from the mRNA of diseased tissue or miRNA (comprising the mRNA or the miRNA that separate from illing tissue) can compare (for example ill mammary tissue sample is to normal galactophore tissue's sample) with the expression intensity of identical entity in the same type healthy tissues.The ratio of these expression intensities has indicated the multiple of genetic expression between described specimen and control sample to change.Perhaps, for example, if vesica is not present in healthy tissues (mammary gland) under normal circumstances, as known in the art, can define the quantity of existing miRNA molecule and the miRNA or the mRNA that separate without the vesica by being derived from healthy tissues by the absolute quantitation method.

In addition, can also show in many ways gene expression profile.Method commonly used be by original fluorescence intensity or ratio arranged in dendriform figure, wherein perpendicular list is shown specimen and is walked crosswise the expression gene.Data are arranged to have a gene of similar express spectra adjacent one another are.The expression ratio that shows each gene with color.For example, ratio can be shown as the blue portion of spectrum lower than 1 (meaning to lower), and ratio is greater than the color that 1 (meaning to raise) can be shown as the red part of spectrum.Commercially available computer software programs can be for showing these data.

Be considered to the mRNA of differential expression or miRNA in ill patient with respect to without the disease individuality, can be express or express not enough.Crossing expression or expressing deficiency is relative term, refers to that the expression amount of finding mRNA or miRNA has detectable difference (exceeding the part of influence of noise in the system for measuring) with respect to some baseline.In this case, the measurement mRNA/miRNA that described baseline is non-disease individuality expresses.Target mRNA/miRNA in diseased cells was to express or express deficiency with respect to the baseline values that uses identical measuring method to obtain.With regard to this one side, the ill change that refers to physical state, this change meeting interrupts or disturbs the correct execution of body function when the not controlled propagation of cell occurs, or has the potential possibility of disturbing the correct execution of human body function.When some aspect of individual genotype or phenotype is consistent with the generation of disease, it is diagnosed as suffers from this disease.Yet, diagnosed or the activity of prognosis comprises to the determining of disease/state issues, such as determining recurrence or the possibility shifted and treating monitoring.In the treatment monitoring, determine that by contrast intragentic expression of for some time the expression of mRNA/miRNA says no and become or whether become the pattern more consistent with healthy tissues, thereby the effect of the given course for the treatment of is made to clinical judgment.

Change to distinguish expression and express not enough level according to the multiple of the intensity measurements of hybridizing micro probe array.Be preferred for carrying out this differentiation 2 * difference, or be less than 0.05 p value.That is, mRNA/miRNA disease/recurrence to normal/non-recurrence cell in before differential expression, the disease cell has been found to produce the intensity that is higher or lower than at least 2 times of described normal cells.Multiple difference is larger, and described gene is more preferred as the application of the instrument of diagnosis or prognosis.The expression level had for the selected mRNA/miRNA of express spectra of the present invention is used the volume production of the background signal of clinical trial instrument to give birth to and the differentiable signal of the signal of normal or non-regulatory gene by having exceeded.

Statistical value can be for being distinguished the mRNA/miRNA of the mRNA/miRNA of regulation and control and non-regulation and control and noise credibly.The statistics test finds that mRNA/miRNA has the most significant difference between the several samples group.Student ' s t verifies as the example of stable statistical test, and it can be for finding the significant difference between two groups.The p value is lower, and gene is more credible at the evidence that does not demonstrate difference between on the same group.However, due to the microarray one-shot measurement more than a kind of mRNA/miRNA, can implement tens thousand of times statistical test simultaneously.Given this, seldom may observe by accident little p value, and can use Sidak correction and random/schedule experiment to make the adjustment for this situation.The evidence that 0.05 p value has significant difference for gene that is less than according to t-check.More believable evidence is less than 0.05 for p value after comprising the factor that Sidak proofreaies and correct.For the great amount of samples in each group, after random/schedule check, to be less than 0.05 be the credible evidence with significant difference to the p value.

In one embodiment, can obtain by the patient by statistically significant quantity mRNA or miRNA (biomarker) expression data; Thereby described data are carried out to linear discriminant analysis and obtain selected biomarker; And the selected biomarker that the expression level of weighting is applied to have the discriminant function factor is so that obtain can be as the predictive model of posterior probability score, form thus can be diagnosed, prognosis, treatment is relevant or the method for the posterior probability score specific biological marking score of physiological status.In addition, other analysis tool also can be for answering identical problem, for example logistic regression and neural net method.

For example, below explanation can be for linear discriminant analysis:

Wherein

I(p _si _dincluded probe sets be take the logarithm of 2 intensity that are the end in)=bracket.The discriminant function of the positive class of d (cp)=disease.D(C _nthe discriminant function of)=disease-negative class.

P ( _cPthe posteriority p value of the positive class of)=disease.

P ( _cNthe posteriority p value of)=disease-negative class.

Multiple other well-known method of pattern recognition is available.Some examples are provided below with reference to document: weighted voting: Golub etc. (1999); SVMs: Su etc. (2001); And Ramaswamy etc. (2001); K is close to algorithm: Ramaswamy (2001); And relation conefficient: (2002) such as van ' t Veer, all documents all are incorporated herein by reference in full.

Can set up the biological marking set hereinafter further described, make the combination of the biomarker in described set there is sensitivity and the specificity of improvement for the combination of the random selection of independent biomarker or biomarker.In one embodiment, can reflect with multiple difference the sensitivity of biological marking set, for example, by showing with respect to the transcript of standard state in morbid state.Specificity can be reflected in the statistical measure that signal that transcript is expressed and the dependency between target condition carry out.For example, standard difference can be used as this tolerance.In considering that one group of biomarker is included in to biological marking set, the little standard difference of expressing in measuring is relevant to higher specificity.In addition, other measurement of variation (for example relation conefficient) also can be for this ability.

The use of the observed value that can be absolute signal difference for another parameter of selecting mRNA/miRNA, wherein said mRNA/miRNA can produce the signal higher than non-regulation and control mRNA/miRNA or noise.The signal that the signal produced by the mRNA/miRNA representation regulated and controled and normal or non-regulatory gene (on the absolute value basis) produce has at least 20% difference.More preferably, this mRNA/miRNA can produce the express spectra that mRNA/miRNA with normal or non-regulation and control has at least 30% difference.

In addition, can also be by be increased to detect and measure miRNA by biological sample, and can use in U.S. Patent No. 7,250,496, U. S. application publication number No.20070292878,20070042380 or 20050222399 reach the method described in the reference wherein quoted measures miRNA, and it is incorporated to this paper in full with way of reference separately.Can be as the U.S. Patent No. that is entitled as " METHODS FOR ASSESSING RNA PATTERNS " 7,888, the 035 assessment microRNA of authorizing on February 15th, 2011, this application is incorporated to this paper in full by reference.

Can in biological marking analysis, use peptide nucleic acid(PNA) (PNA), its new kind that is the nucleic acid analogue, wherein phosphoric acid-sugared polynucleotide main chain is replaced by flexible false peptide polymer.PNA can have highly resistant in complementary RNA and DNA sequence dna and for the degraded of nuclease and proteolytic enzyme with high-affinity and specific hybrid.Peptide nucleic acid(PNA) (PNA) is the new kind of noticeable probe, and it has the cytogenetic application that human chromosomal is carried out the quick in situ evaluation and detects copy number variation (CNV).Polychrome peptide nucleic acid(PNA)-fluorescence in situ hybridization (PNA-FISH) scheme has been described imbalance and the infectious diseases relevant for the identification of several people CNV.PNA can also be as the instrument of molecular diagnosis for the radionuclide with cancer target-PNA-peptide mosaic non-invasively measuring carinogenicity mRNA.The method of using PNA is at Pellestor F etc., Curr Pharm Des.2008; 14 (24): 2439-44, Tian X etc., Ann N Y Acad Sci.2005 November; 1059:106-44, Paulasova P and Pellestor F, Annales de G é n é tiqu e, 47 (2004) 349-358, Stender H.Expert Rev Mol Diagn.2003 September; 3 (5): 649-55. summary, Vigneault etc., Nature Methods, have further description in 5 (9), 777-779 (2008), and each reference all is incorporated herein by reference in full.These methods can be separated for screening the genetic material obtained from vesica.When the specific vesica of cell source is applied to these technology, they can be for the identification of the given molecular signal directly related with cell source.

Can carry out mutation analysis to mRNA and DNA (comprising those that identified by vesica).For the target of originating for RNA or the mutation analysis of biomarker, described RNA (mRNA, miRNA or other RNA) can be reversed record to be become cDNA and is checked order subsequently or analyze, checked order and measured such as the SNP for known (for example, by the Taqman snp analysis) or single nucleotide mutation, thereby and observing and insert or delete and determine the sudden change existed in described cell source with order-checking.The probe amplification of multiple join dependency (MLPA) can alternatively be identified the purpose of CNV for the specific target areas little.For example, once obtain total RNA by the colorectal carcinoma specificity vesica separated, can synthesize cDNA, and the Auele Specific Primer of the exon 2 of KRAS gene and 3 codon 12,13 that can comprise the KRAS gene for amplification and these two exons of 61.Can be for Big Dye Terminator sequential analysis on ABI 3730 for the same primers as of pcr amplification, thus the sudden change in the exon 2 and 3 of KRAS identified.The resistance as Cetuximab and Victibix to medicine is given in sudden change in known these codons.The method of implementing mutation analysis is at Maheswaran S etc., July 2 (10.1056/NEJMoa0800668) in 2008 and Orita, and M etc., PNAS 1989, (86): in 2766-70, describe to some extent, each document all is incorporated herein by reference in full.

Other method of implementing mutation analysis comprises the miRNA order-checking.The application of evaluation and miRNA somatotype can and be used capillary DNA order-checking or " next generation " sequencing technologies to implement by clone technology.The miRNA that current available novel sequencing technologies allows to identify low abundance miRNA or show the moderate differential expression between sample, it may not detected by the method based on hybridization.These new sequencing technologies are included in Nakano etc. 2006, Nucleic Acids Res.2006; 34:D731-D735.doi:10.1093/nar/gkj077 described in extensive parallel signature order-checking (MPSS) method, Margulies etc. 2005, Nature.2005; The Nat.Genet.2006b such as the Roche/454 platform described in 437:376-380 or Berezikov; Illumina order-checking platform described in 38:1375-1377, each document all is incorporated herein by reference in full.

For other method of determining the biological marking, comprise by allele-specific PCR (it comprise specific primer with for increase and distinguish two allelotrope of gene simultaneously), single strand conformation polymorphism (SSCP) (its fine difference related to based on sequence carries out electrophoretic separation to strand Nucleotide) and DNA and RNA is fit measures the biological marking.DNA and RNA are fit is short oligonucleotide sequence, this sequence can be according to its affinity with height in conjunction with the ability of specific molecular by with selecting in hangar.Use fit method at Ulrich H etc., Comb Chem High Throughput Screen.2006 September; 9 (8): 619-32, Ferreira CS etc., Anal Bioanal Chem.2008 February; 390 (4): 1039-50, Ferreira CS etc., Tumour Biol.2006; 27 (6): in 289-301, describe to some extent, each document all is incorporated herein by reference in full.

Can also use fluorescence in situ hybridization (FISH) technology to carry out the detection of biological mark.With FISH detect and locate specific dna sequence, in tissue sample the specific mRNA in location or the method for identifying chromosome abnormalty at Shaffer DR etc., Clin Cancer Res.2007 April 1; 13 (7): 2023-9, Cappuzo F etc., Journal of Thoracic Oncology, the 2nd volume, the 5th phase, in May, 2007, Moroni M etc., Lancet Oncol.2005 May; 6 (5): in 279-86, describe to some extent, each document all is incorporated herein by reference in full.

Provided the explanatory view of analyzing vesica colony for its useful load in Figure 63 E.In one embodiment, method of the present invention comprises the sign phenotype, and thereby it characterizes described phenotype (6332) by catching vesica (6330) and measuring the level (6331) of the microRNA material that wherein comprised complete.

The biological marking that comprises circulating biological mark or vesica can comprise the wedding agent for it.Described wedding agent can be DNA, RNA, fit, monoclonal antibody, polyclonal antibody, Fab, Fab ', single-chain antibody, synthetic antibody, fit (DNA/RNA), class peptide, zDNA, peptide nucleic acid(PNA) (PNA), lock nucleic acid (LNA), lectin, synthetic or naturally occurring chemical compound (including but not limited to medicine and labelled reagent).

According to mentioned above, wedding agent can be by combining with the component of vesica for separating of or detect described vesica.Described wedding agent can, for detection of vesica, for example detect the specific vesica of cell source.One or more wedding agents himself can form the wedding agent spectrum, and it provides the biological marking of vesica.One or more wedding agents can be selected from Fig. 2.For example, if use 2 kinds, 3 kinds or 4 kinds of wedding agents to detect at the vesica Difference test of heterogeneous vesica colony or in separating or separate vesica colony, provide the biological marking of described specific vesica colony for the particular combination agent spectrum of described vesica colony.

As illustrative example, can use one or more wedding agents to detect the vesica for characterizing cancers, described wedding agent includes but not limited to PSA, PSMA, PCSA, PSCA, B7H3, EpCam, TMPRSS2, mAB 5D4, XPSM-A9, XPSM-A10, Gal-3, CD62L, half lactadherin-1 or E4 (IgG2a κ), or their any combination.

Described wedding agent can also be for general vesica biomarker, such as " house keeping protein " or antigen.Described biomarker can be CD9, CD63 or CD81.For example, described wedding agent can be the antibody for CD9, CD63 or CD81.Described wedding agent can also be for other oroteins, such as tissue specificity or cancer specific vesica.Described wedding agent can be for PCSA, PSMA, EpCam, B7H3 or STEAP.Described wedding agent can be for DR3, STEAP, epha2, TMEM211, MFG-E8, annexin V, TF, unc93A, A33, CD24, NGAL, EpCam, MUC17, TROP2 or TETS.For example, described wedding agent can be for the antibody of PCSA, PSMA, EpCam, B7H3, DR3, STEAP, epha2, TMEM211, MFG-E8, annexin V, TF, unc93A, A33, CD24, NGAL, EpCam, MUC17, TROP2 or TETS or fit.

Usually, various protein not on average or equably is distributed on the vesica shell.For example, referring to Figure 64, it has shown the schematic diagram of protein expression mode.The specific protein of vesica is usually more common, and the protein of cancer specific is more rare.In some embodiments, catching with the more common lower protein of cancer specific of vesica completes, such as one or more house keeping proteins or antigen or general vesica antigen (as four transmembrane proteins), and use one or more cancer specific biomarkers and/or one or more cell source specific biological marks at detecting stage.In another embodiment, use one or more cancer specific biomarkers and/or one or more cell source specific biological marks to be caught, and use one or more house keeping proteins or antigen or general vesica antigen (as four transmembrane proteins) to be detected.In embodiments, use identical biomarker for catching with detecting.Can use the different wedding agents for identical biomarker, such as the antibody of the different epi-positions of conjugated antigen or fit.

By any ordinary method known in the art or according to described herein, can identify other the Cell binding companion body or wedding agent, and it can be used as diagnosis, prognosis and treatment Research of predicting markers in addition.

As illustrative example, can detect the vesica of analyzing for lung cancer with one or more wedding agents, wherein said wedding agent includes but not limited to the fit HCA 12 of SCLC specificity, the fit HCC03 of SCLC specificity, the fit HCH07 of SCLC specificity, the fit HCH01 of SCLC specificity, A-p50 is fit (NF-KB), Cetuximab, Victibix, rhuMAb-VEGF, L19Ab, F16Ab, anti-CD45 (anti-ICAM-1, aka UV3) or L2G7Ab (anti-HGF) or their any combination.

Can detect for characterizing the vesica of colorectal carcinoma with one or more wedding agents, wherein said wedding agent includes but not limited to that angiogenic factors 2 specificitys are fit, beta-catenin is fit, TCF1 is fit, anti-Derlin1 ab, anti-RAGE, mAbgb3.1, Gal-3, Cetuximab, Victibix, horse trastuzumab, rhuMAb-VEGF, Mac-2 or their any combination.

Can detect for characterizing the vesica of adenoma to colorectal carcinoma (CRC) with one or more wedding agents, wherein said wedding agent includes but not limited to complement C3, is rich in the glycoprotein of Histidine, prokinin-1 or Gal-3 or their any combination.

Can detect the adenoma that has a low dysplasia for the sign vesica to adenoma with high grade dysplasia with wedding agent, wherein said wedding agent is such as but not limited to Gal-3 or this contrast is had to any combination of specific wedding agent.

Can detect for characterizing the vesica of CRC to standard state with one or more wedding agents, wherein said wedding agent includes but not limited to anti-ODC mAb, anti-CEA mAb or Mac-2 or their any combination.

Can detect for characterizing the vesica of prostate cancer with one or more wedding agents, wherein said wedding agent includes but not limited to PSA, PSMA, TMPRSS2, mAB 5D4, XPSM-A9, XPSM-A10, Gal-3, CD62L, half lactadherin-1 or E4 (IgG2a κ) or their any combination.

Can detect for characterizing melanomatous vesica with one or more wedding agents, wherein said wedding agent include but not limited to Sibutramine Hydrochloride wood monoclonal antibody (Tremelimumab) (anti-CTLA 4), easily Puli's monoclonal antibody (Ipilimumumab) (anti-CTLA 4), CTLA-4 is fit, the STAT-3 peptide is fit, half lactadherin-1, Gal-3 or PNA or their any combination.

Can detect for characterizing the vesica of carcinoma of the pancreas with one or more wedding agents, wherein said wedding agent includes but not limited to that H38-15 (anti-HGF) is fit, H38-21 (anti-HGF) is fit, horse trastuzumab, Cetuximab (Cetuximanb) or rhuMAb-VEGF or their any combination.

Can detect for characterizing the vesica of the cancer of the brain with one or more wedding agents, wherein said wedding agent includes but not limited to fit III.1 (pigpen) and/or TTA1 (tenascin-C) is fit or their any combination.

Can detect for characterizing psoriatic vesica with one or more wedding agents, wherein said wedding agent includes but not limited to CD62L, ICAM-1, VLA-4, VCAM-1, α E β 7 or their any combination.

Can detect for characterizing the vesica of cardiovascular disorder (CVD) with one or more wedding agents, wherein said wedding agent includes but not limited to RB007 (factors IX A is fit), ARC1779 (anti-VWF) fit or LOX1 or their any combination.

Can detect for characterizing the vesica of hematologic malignancies with one or more wedding agents, wherein said wedding agent includes but not limited to anti-CD20 and/or anti-CD52 or their any combination.

Can detect for characterizing the vesica of B cell lymphocytic leukemia with one or more wedding agents, wherein said wedding agent includes but not limited to Rituximab, alemtuzumab, Apt48 (BCL6), R0-60, D-R15-8 or their any combination.

Can detect for characterizing the vesica of B-cell lymphoma with one or more wedding agents, wherein said wedding agent includes but not limited to ibritumomab tiuxetan, tositumomab, anti-CD20 antibodies, alemtuzumab, markon's former times monoclonal antibody, anti-cd40 antibody, epratuzumab, Shandong former times monoclonal antibody, Hu1D10, Gal-3 or Apt48 or their any combination.

Can detect for characterizing the vesica of burkitt's lymphoma with one or more wedding agents, wherein said wedding agent includes but not limited to that TD05 is fit, IgM mAB (38-13) or their any combination.

Can detect for characterizing the vesica of cervical cancer with one or more wedding agents, wherein said wedding agent includes but not limited to half lactadherin-9 and/or HPVE7 is fit or their any combination.

Can detect for characterizing the vesica of carcinoma of endometrium with one or more wedding agents, wherein said wedding agent includes but not limited to half lactadherin-1 or carcinoma of endometrium is had to any combination of specific wedding agent.

Can detect for characterizing the vesica of neck cancer with one or more wedding agents, wherein said wedding agent includes but not limited to (111) In-cMAb U36, anti-LOXL4, U36, BIWA-1, BIWA-2, BIWA-4 or BIWA-8 or their any combination.

Can detect for characterizing the vesica of IBD with one or more wedding agents, wherein said wedding agent includes but not limited to ACCA (anti-glycan Ab), ALCA (anti-glycan Ab) or AMCA (anti-glycan Ab) or their any combination.

Can detect for characterizing the vesica of diabetes with one or more wedding agents, wherein said wedding agent includes but not limited to that RBP4 is fit or diabetes is had to any combination of specific wedding agent.

Can detect for characterizing the vesica of fibromyalgia with one or more wedding agents, wherein said wedding agent includes but not limited to L-selection albumen or fibromyalgia is had to any combination of specific wedding agent.

Can detect for characterizing the vesica of multiple sclerosis (MS) with one or more wedding agents, wherein said wedding agent includes but not limited to natalizumab (Tysabri) or MS is had to any combination of specific wedding agent.

In addition, can detect for characterizing rheumatismal vesica with one or more wedding agents, wherein said wedding agent includes but not limited to Rituximab (anti-CD20Ab) and/or keliximab (anti-CD4Ab) or rheumatosis is had to any combination of specific wedding agent.

Can detect for characterizing the vesica of alzheimer's disease with one or more wedding agents, wherein said wedding agent includes but not limited to that TH14-BACE1 is fit, S10-BACE1 is fit, anti-A β, bar pearl monoclonal antibody (AAB-001)-Elan, LY2062430 (anti-amyloid beta Ab)-EliLilly or BACE1-antisense or their any combination.

Can detect for characterizing the vesica of prion-specific disease with one or more wedding agents, wherein said wedding agent includes but not limited to that rhuPrP (c) is fit, DP7 is fit, sulfo-is fit 97, SAF-93 is fit, 15B3 (anti-PrPSc Ab), monoclonal anti PrPSc antibody P1: 1,1.5D7,1.6F4 Abs, mab 14D3, mab 4F2, mab 8G8 or mab 12F10 or their any combination.

Can detect for characterizing pyemic vesica with one or more wedding agents, wherein said wedding agent includes but not limited to HA-1AmAb, E-5mAb, TNF-α MAb, Afelimomab or CD62L or their any combination.

Can detect for characterizing schizoid vesica with one or more wedding agents, wherein said wedding agent includes but not limited to L-selection albumen and/or N-CAM or schizophrenia is had to any combination of specific wedding agent.

Can detect for characterizing the vesica of dysthymia disorders with one or more wedding agents, wherein said wedding agent includes but not limited to GPIb or dysthymia disorders is had to any combination of specific wedding agent.

Can detect for characterizing the vesica of GIST with one or more wedding agents, wherein said wedding agent includes but not limited to ANTI-DOG1 Ab or GIST is had to any combination of specific wedding agent.

Can detect for characterizing the vesica of the esophageal carcinoma with one or more wedding agents, wherein said wedding agent includes but not limited to the CaSR wedding agent or the esophageal carcinoma is had to any combination of specific wedding agent.

Can detect for characterizing the vesica of cancer of the stomach with one or more wedding agents, wherein said wedding agent includes but not limited to calpain nCL-2 wedding agent and/or drebrin wedding agent or cancer of the stomach is had to any combination of specific wedding agent.

Can detect for characterizing the vesica of COPD with one or more wedding agents, wherein said wedding agent includes but not limited to CXCR3 wedding agent, CCR5 wedding agent, CXCR6 wedding agent or COPD is had to any combination of specific wedding agent.

Can detect for characterizing the vesica of asthma with one or more wedding agents, wherein said wedding agent includes but not limited to VIP wedding agent, PACA wedding agent, CGRP wedding agent, NT3 wedding agent, YKL-40 wedding agent, S-nitrosothiol, SCCA2 wedding agent, PAI wedding agent, amphiregulin wedding agent or periostin wedding agent or asthma is had to any combination of specific wedding agent.

Can detect for characterizing the vesica of vulnerable plaque with one or more wedding agents, wherein said wedding agent includes but not limited to Gd-DTPA-g-mimRGD (α v β 3 integrin binding peptides) or MMP-9 wedding agent or vulnerable plaque is had to any combination of specific wedding agent.

Can detect for characterizing the vesica of ovarian cancer with one or more wedding agents, wherein said wedding agent includes but not limited to (90) Y-muHMFG1 wedding agent and/or OC125 (anti-CA 125 antibody) or ovarian cancer is had to any combination of specific wedding agent.

Described wedding agent also can be for general vesica biomarker, such as " house keeping protein " or antigen.Described biomarker can be CD9, CD63 or CD81.For example, described wedding agent can be the antibody for CD9, CD63 or CD81.Described wedding agent can also be for other oroteins, such as for prostate specific or vesica cancer specific.Described wedding agent can be for PCSA, PSMA, EpCam, B7H3 or STEAP.For example, described wedding agent can be the antibody for PCSA, PSMA, EpCam, B7H3 or STEAP.

In addition, by traditional method known in the art or according to described herein, can identify other the Cell binding companion body or wedding agent, and it can be used as diagnosis, prognosis and treatment Research of predicting markers in addition.

The biological marking for prostate cancer, GI cancer and ovarian cancer

As described herein, the biological marking that comprises the circulating biological mark can be used for characterizing cancers.These chapters and sections have provided the list of non-limit of the biomarker of the part that can be used as the biological marking (for example,, for prostate gland, GI or ovary cancer).In some embodiments, described circulating biological mark is associated with vesica or vesica colony.For example, the circulating biological mark associated with vesica can be used for catching and/or detects vesica or vesica colony.These chapters and sections have provided the list of non-limit of the biomarker of the part that can be used as the biological marking (for example,, for prostate gland, GI or ovary cancer).

Should be appreciated that the biomarker that this paper provides can be used in the biological marking of Other diseases (as the cancer of other proliferative imbalance and other cell or tissue origin).For example, the conversion in various different cell types may cause due to common event, as the sudden change of p53 or other tumor-inhibiting factor.The biological marking that comprises cell source biomarker and biomarker for cancer can be used for the characteristic of further assessment of cancer.The biomarker of metastatic cancer can be used to assess metastatic cancer together with the cell source biomarker.Comprise Dawood for these biomarkers of the present invention, Novelbiomarkers of metastatic cancer, Exp Rev Mol Diag in July, 2010, the 10th volume, the 5th chapter, those biomarkers in the 581-590 page, this publication is incorporated to this paper in full by reference.

It is that raise, that lower or unconverted mark that the biological marking of the present invention can comprise according to reference.Only for purposes of illustration, if described reference is normal specimens, at described experimenter's the biological marking, compares the described biological marking in unconverted situation with described reference and can show that described experimenter is normal.Perhaps, the described biological marking can comprise nucleic acid or the aminoacid sequence of sudden change, thereby makes between normal reference and ill sample in the described biological marking level of composition identical.In another case, described reference can be the cancer sample, thereby means cancer in the situation that described experimenter's the biological marking is substantially similar to described this experimenter's of reference the biological marking.Described experimenter's the biological marking can comprise simultaneously compares the composition that upper mediation is lowered with described reference.Only for purpose of explanation, if described reference is normal specimens, the biological marking of cancer can comprise the oncogene of rise and the tumor suppressor gene of downward simultaneously.The vesica mark also can be expressed in various varying environment allowances below nominal size strange land.For example, with non-cancer vesica, compare, four transmembrane proteins can be crossed and express in the cancer vesica, and compare with the cancer vesica, and MFG-E8 can cross and express in non-cancer vesica.

Prostate cancer

Prostate specific antigen (PSA) is the protein produced by prostatic cell.PSA exists a small amount of in normal male serum, and it is in the situation that there are usually rising to some extent in prostate cancer (PCa) and the imbalance of other prostate gland.The blood test that is used for measuring PSA is at present for the examination prostate cancer, but its validity also is under suspicion.For example, prostate gland infection, stimulation, benign prostatic hyperplasia (BPH), digital rectal examination (DRE) and ejaculation recently can cause the PSA level to improve, thereby the generation false positive results, this may cause unnecessary prostate gland tissue biopsy and the misery of following.BPH is the common cause that the PSA level rises.PSA can show whether prostate gland some problem occurs, but it can not distinguish BPH and PCa effectively.PCA3 (it is found that it is the transcript that prostate cancer cell is crossed expression) is considered to have slightly high specificity for PCa, but this depends on for the cutoff of PSA and PCA3 and the colony of studying.

The invention provides the circulating biological mark, it can be used for distinguishing BPH and PCa.Thereby biomarker group is assessed BPH is distinguished with PCa.Described group can be used for detecting the vesica that presents the particular surface mark.In some embodiments, described surface marker comprises one or more in BCMA, CEACAM-1, HVEM, IL-1 R4, IL-10 Rb and Trappin-2.Can analyze also subsequently for distinguishing BPH and PCa the level of the biomarker in the vesica that is derived from blood sample.

In yet another aspect, microRNA (miR) is for distinguishing BPH and prostate cancer.Described miR can directly separate and/or can analyze described vesica for the miR useful load comprised the vesica that is derived from patient's sample from patient's sample.Described sample can be body fluid, comprises seminal fluid, urine, blood, serum or blood plasma.Described sample also can comprise tissue or tissue biopsy's sample.Can utilize a large amount of different methods for detection of according to miR as herein described.In some embodiments, the array of miR group is for detect the expression of multiple miR simultaneously.For example, Exiqon mIRCURY LNA microRNA PCR system group (Exiqon, Inc., Woburn, MA) can be used for this classification.The miR that distinguishes BPH and PCa compares with the PCa sample and can in the BPH sample, cross and express, and it includes but not limited to one or more in hsa-miR-329, hsa-miR-30a, hsa-miR-335, hsa-miR-152, hsa-miR-151-5p, hsa-miR-200a and hsa-miR-145.Perhaps, the miR that distinguishes BPH and PCa can cross and express with respect to the BPH sample in the PCa sample, it includes but not limited to hsa-miR-29a, hsa-miR-106b, hsa-miR-595, hsa-miR-142-5p, hsa-miR-99a, hsa-miR-20b, hsa-miR-373, hsa-miR-502-5p, hsa-miR-29b, hsa-miR-142-3p, hsa-miR-663, hsa-miR-423-5p, hsa-miR-15a, hsa-miR-888, hsa-miR-361-3p, hsa-miR-365, hsa-miR-10b, hsa-miR-199a-3p, hsa-miR-181a, hsa-miR-19a, hsa-miR-125b, hsa-miR-760, hsa-miR-7a, hsa-miR-671-5p, hsa-miR-7c, one or more in hsa-miR-1979 and hsa-miR-103.

Can be assessed and itself and reference level are compared to the miR expressed with checkout discrepancy the expression level of one or more above-mentioned miR, the result output of diagnosis, prognosis or treatment diagnosis is provided thus.Described reference level can be the level that is derived from the miR in the normal patient exosome of (as do not suffered from the patient of prostatosis).Therefore, the differential expression that one or more miR are different from described reference level can mean that described sample is different from normally, for example, comprises BPH or PCa.Described reference level can be the level that is derived from the miR in BPH patient's exosome.Therefore, the differential expression that one or more miR are different from described reference level can mean that described sample is different from BPH, for example, comprises normal or PCa.Described reference level can be the level that is derived from the miR in PCa patient's exosome.Therefore, the differential expression that one or more miR are different from described reference level can mean that described sample is different from PCa, for example, comprises normal or BPH.

In some embodiments, the level of one or more miR in specimen is interrelated with the level of identical miR in the reference sample, and the result output of diagnosis, prognosis or treatment diagnosis is provided thus.The described miR level that can comprise the one or more samples with BPH, PCa with reference to sample, or can be from the normal person who does not there is BPH or PCa.When the level of one or more miR in specimen and normal reference level are the most relevant, described specimen can classify as normally.When the level of one or more miR in described specimen and BPH reference level are the most relevant, described specimen can classify as BPH.When the level of one or more miR in specimen and PCa reference level are the most relevant, described specimen can classify as PCa.

The biological marking can be used for characterizing prostate cancer.As described above, the biological marking of prostate cancer can comprise and the Cancer-Related wedding agent of prostate gland (for example, shown in Fig. 2); And one or more other biomarkers, for example, shown in Figure 19.For example, the biological marking of prostate cancer can comprise the wedding agent for PSA, PSMA, TMPRSS2, mAB 5D4, XPSM-A9, XPSM-A10, Gal-3, E-half lactadherin, half lactadherin-1, E4 (IgG2a κ) or their any combination; And one or more other biomarkers, for example one or more miRNA, one or more DNA, one or more and Cancer-Related other peptide of prostate gland, protein or antigen, shown in Figure 19.

The biological marking of prostate cancer can comprise and the Cancer-Related antigen of prostate gland, for example, shown in Fig. 1, and one or more other biomarkers, for example, shown in Figure 19.The biological marking of prostate cancer can comprise and Cancer-Related one or more antigens of prostate gland, such as but not limited to KIA1, complete fibronectin, PSA, TMPRSS2, FASLG, TNFSF10, PSMA, NGEP, IL-7RI, CSCR4, CysLT1R, TRPM8, Kv1.3, TRPV6, TRPM8, PSGR, MISIIR or their any combination.The biological marking of prostate cancer can comprise one or more aforesaid antigens and one or more other biomarkers, such as but not limited to miRNA, mRNA, DNA or their any combination.

The biological marking of prostate cancer can also comprise one or more and the Cancer-Related antigen of prostate gland, such as but not limited to KIA1, complete fibronectin, PSA, PCA3, TMPRSS2, TMPRSS2-ERG, FASLG, TNFSF10, PSMA, NGEP, IL-7RI, CSCR4, CysLT1R, TRPM8, Kv1.3, TRPV6, TRPM8, PSGR, MISIIR or their any combination, and one or more miRNA biomarkers, such as but not limited to miR-202, miR-210, miR-296, miR-320, miR-370, miR-373, miR-498, miR-503, miR-184, miR-198, miR-302c, miR-345, miR-491, miR-513, miR-32, miR-182, miR-31, miR-26a-1/2, miR-200c, miR-375, miR-196a-1/2, miR-370, miR-425, miR-425, miR-194-1/2, miR-181a-1/2, miR-34b, let-7i, miR-188, miR-25, miR-106b, miR-449, miR-99b, miR-93, miR-92-1/2, miR-125a, miR-141, let-7a, let-7b, let-7c, let-7d, let-7g, miR-16, miR-23a, miR-23b, miR-26a, miR-92, miR-99a, miR-103, miR-125a, miR-125b, miR-143, miR-145, miR-195, miR-199, miR-221, miR-222, miR-497, let-7f, miR-19b, miR-22, miR-26b, miR-27a, miR-27b, miR-29a, miR-29b, miR-30_5p, miR-30c, miR-100, miR-141, miR-148a, miR-205, miR-520h, miR-494, miR-490, miR-133a-1, miR-1-2, miR-218-2, miR-220, miR-128a, miR-221, miR-499, miR-329, miR-340, miR-345, miR-410, miR-126, miR-205, miR-7-1/2, miR-145, miR-34a, miR-487 or let-7b or their any combination.

The biological marking of prostate cancer also can comprise one or more circulating biological marks, such as the microRNA relevant to prostate cancer, it comprises and is described in Brase etc., Circulating miRNAs are correlated with tumor progression in prostate cancer.Int J Cancer.2011 February 1; 128 (3): 608-16; Wach etc., MiRNA profiles of prostate carcinoma detected by multi-platform miRNA screening.Int J Cancer.2011 .doi:10.1002/ijc.26064 on March 11; Gordanpour etc., miR-221 Is Down-regulated in TMPRSS2:ERG Fusion-positive Prostate Cancer.Anticancer Res.2011 February; 31 (2): 403-10; Hagman etc., miR-34c is downregulated in prostate cancer and exerts tumor suppressive functions.Int J Cancer.2010 December 15; 127 (12): 2768-76; Sun etc., miR-99 Family of MicroRNAs Suppresses the Expression of Prostate-Specific Antigen and Prostate Cancer Cell Proliferation.Cancer Res.2011 February 15; 71 (4): 1313-24; Bao etc., Polymorphisms inside MicroRNAs and MicroRNA Target Sites Predict Clinical Outcomes in Prostate Cancer Patients Receiving Androgen-Deprivation Therapy.Clin Cancer Res.2011 February 15; 17 (4): 928-936; Moltzahn etc., Microfluidic-based multiplex qRT-PCR identifies diagnostic and prognostic microRNA signatures in the sera of prostate cancer patients.Cancer Res.2011 January 15; 71 (2): 550-60; Carlsson etc., Validation of suitable endogenous control genes for expression studies of miRNA in prostate cancer tissues.Cancer Genet Cytogenet.2010 October 15; 202 (2): 71-75; Zhang etc., Serum miRNA-21:elevated levels in patients with metastatic hormone-refractory prostate cancer and potential predictive factor for the efficacy of docetaxel-based chemotherapy.Prostate.2011 February 15; 71 (3): 326-31; Majid etc., MicroRNA-205-directed transcriptional activation of tumor suppressor genes in prostate cancer.Cancer.2010 December 15; 116 (24): 5637-49; Kojima etc., MiR-34a attenuates paclitaxel-resistance of hormone-refractory prostate cancer PC3 cells through direct and indirect mechanisms.Prostate.2010 October 1; 70 (14): 1501-12; Lewinshtein etc., Genomic predictors of prostate cancer therapy outcomes.Expert Rev Mol Diagn.2010 July; 10 (5): the microRNA in 619-36; Each publication is incorporated in full by reference.

In addition, the miRNA of the biological marking of prostate cancer can for PFKFB3, RHAMM (HMMR), cDNA FLJ42103, ASPM, CENPF, NCAPG, androgen receptor, EGFR, HSP90, SPARC, DNMT3B, GART, MGMT, SSTR3, TOP2B or their the interactional miRNA of any combination, for example those shown in described herein and Figure 60.Described miRNA can be also miR-9, miR-629, miR-141, miR-671-3p, miR-491, miR-182, miR-125a-3p, miR-324-5p, miR-148B, miR-222 or their any combination.

The biological marking of prostate cancer can comprise one or more and the Cancer-Related antigen of prostate gland, such as but not limited to KIA1, complete fibronectin, PSA, TMPRSS2, FASLG, TNFSF10, PSMA, NGEP, IL-7RI, CSCR4, CysLT1R, TRPM8, Kv1.3, TRPV6, TRPM8, PSGR, MISIIR or their any combination; And one or more other biomarkers, such as but not limited to described miRNA, mRNA (such as but not limited to AR or PCA3), snoRNA (such as but not limited to U50) or their any combination before.

In addition, the described biological marking can also comprise one or more gene fusion, for example ACSL3-ETV1, C15ORF21-ETV1, FLJ35294-ETV1, HERV-ETV1, TMPRSS2-ERG, TMPRSS2-ETV1/4/5, TMPRSS2-ETV4/5, SLC5A3-ERG, SLC5A3-ETV1, SLC5A3-ETV5 or KLK2-ETV4.

Can be separated, be analyzed or the two vesica with the Cancer-Related antigen of one or more and prostate gland for one or more miRNA, thereby provide diagnosis, prognosis or treatment diagnosis spectrum, for example further feature of the effect of the stage of cancer, cancer or cancer.Perhaps, can be by sample direct analysis vesica, before being analyzed for one or more miRNA or with the Cancer-Related antigen of prostate gland, described vesica is not purified or concentrated thus.

As shown in Figure 68, the biological marking of prostate cancer can comprise EpCam, CD63, CD81, CD9 or their any combination of analyzing vesica.The biological marking of described prostate cancer can comprise detection EpCam, CD9, CD63, CD81, PCSA or their any combination.For example, the biological marking of described prostate cancer can comprise EpCam, CD9, CD63 and CD81 or PCSA, CD9, CD63 and CD81 (for example, referring to Figure 70 A).The biological marking of described prostate cancer can also comprise PCSA, PSMA, B7H3 or their any combination (for example, referring to Figure 70 B).

In addition, the situation that is less than multiple biomarker with assessment is compared, and assessing multiple biomarker can provide the sensitivity of increase, specificity or strength of signal.For example, with independent assessment PMSA or B7H3, compare, assessment PSMA and B7H3 can provide the sensitivity of increase in detection.With independent assessment CD9 or CD63, compare, assessment CD9 and CD63 can provide the sensitivity of increase in detection.In one embodiment, one or more following biomarker: EpCam, CD9, PCSA, CD63, CD81, PSMA, B7H3, PSCA, ICAM, STEAP and EGFR have been detected.In another embodiment, EpCam+, CK+, CD45-vesica have been detected.

Can also characterize prostate cancer according to meeting at least 1,2,3,4,5,6,7,8,9 or 10 standard.For example, can use multiple different standard: 1) whether from the amount of the vesica in experimenter's sample higher than reference point; 2) whether the amount of the vesica in prostatic cell source higher than reference point; And 3) amount of vesica that whether has one or more cancer specific biomarkers is higher than reference point, and described experimenter is diagnosed as and suffers from prostate cancer.Described method may further include qualitative contrast and measures.

In another embodiment, one or more biological markings of vesica are used to making diagnosis between normal prostatic and prostate cancer or between normal prostatic, BPH and PCa.Suitable biomarker arbitrarily disclosed herein can be used for identification PCa.In some embodiments, one or more trapping agents commonly used for biomarker (or catch biomarker, detected or the biomarker of combination by trapping agent) can be used for from one or more vesicas of the analyte capture from the experimenter.

The prostate specific biomarker can be used for identifying the prostate specific vesica.Biomarker for cancer can be used for identifying the cancer specific vesica.In some embodiments, one or more in CD9, CD81 and CD63 are used as catching biomarker.In some embodiments, PCSA is used as the prostate gland biomarker.In some embodiments, described one or more biomarker for cancer comprise one or more in EPCam and B7H3.The other biomarker of PCa and normal phase differentiation can be comprised to ICAM1, EGFR, STEAP1 and PSCA.

In some embodiments, in the experimenter, the method for evaluation prostate cancer comprises: (a) use trapping agent to catch vesica colony in the sample from described experimenter; (b) measure the level of one or more biomarker for cancer in described vesica colony; (c) measure the level of one or more prostate gland biomarkers in described vesica colony; If, and (d) level of the level of described one or more biomarker for cancer and one or more prostate gland biomarkers meets predetermined threshold value, described experimenter is accredited as and suffers from prostate cancer.In some embodiments, described trapping agent comprises one or more wedding agents for CD9, CD81 and CD63.In some embodiments, described one or more prostate gland biomarkers comprise PCSA and/or PSMA.In some embodiments, described one or more biomarker for cancer comprise one or more in EPCam and B7H3.In other embodiments, the wedding agent of described one or more prostate glands and/or biomarker for cancer is as trapping agent, and the wedding agent of described one or more general vesica marks is as detection agent.In some embodiments, described predetermined threshold value comprises the observed value of detectable.For example, detectable can be that fluorescence part and described value can be the luminous values of this part.

In another embodiment, determine the prognosis of prostate cancer by the expression that detects EpCam, CK (cytokeratin) and/or CD45.In one embodiment, detecting or detecting EpCam and CK and lower or poor prognosis is provided while not having (vesica that is EpCam+, CK+, CD45-) to the detection of CD45 with high level.

Can use method of the present invention to distinguish stage and the degree of prostate cancer.Prostate cancer be that cancer is diffused into to the process that the risk outside prostate gland is sorted out by stages.This diffusion is relevant to the possibility of curing by topical therapeutic (such as operation or radiation).The information of considering in this prognosis classification is based on clinical and Pathologic factors, and it comprises physical examination, imaging research, blood test and/or tissue biopsy.

Issued by american cancer federation (American Joint Committee on Cancer) for the most frequently used scheme of prostate cancer being carried out by stages, and be called the TNM system.Degree, the transfer of the size of described TNM system evaluation tumour, the lymphoglandula involved, and consider the cancer grade.The same with multiple other cancer, these cancers are grouping (for example I-IV phase) on schedule usually.Generally, I phase disease be when the prostatitis glandular tissue due to other reason (such as benign prostate cancer hyperplasia) while obtaining occasionally in the cancer in the small portion sample, and cell is similar to normal cell nearly and body of gland is normal for the doigte of check.Interim at II, involve more prostate gland and can feel agglomerate in body of gland.Interim at III, tumour diffuses to whole prostatic utriculus and can arrive agglomerate in the surface feel of body of gland.In IV phase disease, tumour has been invaded proximity structure, or has diffused to lymphoglandula or other organ.

Whitmore-Jewett is the another kind of staging system be of little use at present by stages.Gleason hierarchy system (Gleason Grading System) is based on entocyte and weave construction from tissue biopsy, and it provides the estimation to the damage potential of this disease and final prognosis.

Described TNM staging system can be used for describing the degree of cancer in subject.T describes the size of described tumour and whether it has invaded adjacent tissue, and N describes the regional nodes involved, and M describes far away the transfer.TNM (UICC) is safeguarded and is used by american cancer federation (AJCC) and FIGO (FIGO) by International Union Against Cancer (International Union Against Cancer).Those skilled in the art understands, and is not that all cancers have TNM classification, for example cancer of the brain.Generally, ((0), 1-4) be determined as size or the direct degree of primary tumo(u)r to T for a, is.N (0-3) refers to the degree of the diffusion of regional nodes: N0 and refers to that there is not tumour cell in regional nodes, and N1 refers to that tumour cell diffuses to the regional nodes of the most contiguous or minority, and N2 refers to that the tumour cell diffusion is between N1 and N3; N3 refers to that tumour cell diffuses to farthest or a large amount of regional nodeses.M (0/1) refers to the existence of transfer: it is not evaluated that MX means transfer far away; M0 means and does not have transfer far away; M1 means transfer and has betided remote organs (exceeding regional nodes).M1 can further describe as follows: M1a means that cancer has been diffused into the lymphoglandula that exceeds regional nodes; M1b means that cancer has diffused to bone; And M1c means that cancer has diffused to other position (no matter whether relating to bone).Also can evaluate other parameter.G (1-4) refers to the grade (that is, they,, for low-grade, are high-grade if they show as poorly differentiated if they show to such an extent that be similar to normal cell) of cancer cells.R (0/1/2) refers to the thoroughness (that is, whether the excision border of cancer-free cell) of operation.L (0/1) refers to and invades in lymphatic vessel.V (0/1) refers to and invades in vein.C (1-4) refers to the correction symbol of the determinacy (characteristic) to V.

Tumor of prostate is used the Gleason points-scoring system to be assessed usually.Described Gleason points-scoring system is based on the micro-tumour pattern of being evaluated by the pathologist when explaining tissue biopsy's sample.While in tissue biopsy, having prostate cancer, described Gleason scoring is the extent of damage according to normal gland tissue structure (that is, the shape of described body of gland, size and differentiation).Classical Gleason points-scoring system has 5 kinds of standard weave's patterns, and it is called tumour " grade " technically.The micrography that this loss of the normal gland structure that caused by cancer is carried out means by grade, and it is the numeral between 1 and 5, and 5 is the poorest grade.1 grade is generally that wherein carcinous prostate gland is similar to the situation of normal prostate tissue closely.Body of gland is little, well-formed's (well-formed) and tight enclosing.In 2 grades, organize the body of gland that still there is the well-formed, but it has more greatly and therebetween more tissue, still have discernible body of gland and organize in 3 grades, but cell is darker.Under high magnification, some cells in 3 grades of samples have broken away from body of gland and have started to invade surrounding tissue.4 grades of samples have the tissue that has hardly discernible body of gland, and many cells are being invaded surrounding tissue.For 5 grades of samples, organize and do not there is discernible body of gland and be generally the cell sheets that spreads all over surrounding tissue.

The miR that distinguishes transitivity and non-metastatic prostate cancer can cross and express with respect to the non-metastatic sample in the transitivity sample.Perhaps, the miR that distinguishes transitivity and non-metastatic prostate cancer can cross and express with respect to the transitivity sample in the non-metastatic sample.Comprise one or more in miR-495, miR-10a, miR-30a, miR-570, miR-32, miR-885-3p, miR-564 and miR-134 for the available miR that distinguishes metastatic prostate cancer.In another embodiment, comprise hsa-miR-375, hsa-miR-452, hsa-miR-200b, hsa-miR-146b-5p, hsa-miR-1296, hsa-miR-17 for the miR that distinguishes metastatic prostate cancer ^*, hsa-miR-100, hsa-miR-574-3p, hsa-miR-20a ^*, hsa-miR-572, hsa-miR-1236, hsa-miR-181a, hsa-miR-937 and hsa-miR-23a ^*in one or more.In another embodiment again, for the available miRN that distinguishes metastatic prostate cancer, comprise hsa-miR-200b, hsa-miR-375, hsa-miR-582-3p, hsa-miR-17 ^*, hsa-miR-1296, hsa-miR-20a ^*, one or more in hsa-miR-100, hsa-miR-452 and hsa-miR-577.For the miR that distinguishes metastatic prostate cancer, can be one or more of miR-141, miR-375, miR-200b and miR-574-3p.

In yet another aspect, microRNA (miR) is for distinguishing cancer and non-cancer sample.Comprise hsa-miR-574-3p, hsa-miR-331-3p, hsa-miR-326, hsa-miR-181a-2 for the available miR that cancer and non-cancer are distinguished ^*, hsa-miR-130b, hsa-miR-301a, hsa-miR-141, hsa-miR-432, hsa-miR-107, hsa-miR-628-5p, hsa-miR-625 ^*, one or more in hsa-miR-497 and hsa-miR-484.In another embodiment, comprise hsa-miR-574-3p, hsa-miR-141, hsa-miR-331-3p, hsa-miR-432, hsa-miR-326, hsa-miR-2110, hsa-miR-107, hsa-miR-130b, hsa-miR-301a and hsa-miR-625 for the available miR that distinguishes cancer and non-cancer ^*in one or more.In another embodiment again, for the available miR that distinguishes cancer and non-cancer, comprise hsa-miR-107, hsa-miR-326, hsa-miR-432, hsa-miR-574-3p, hsa-miR-625 ^*, hsa-miR-2110, hsa-miR-301a, hsa-miR-141 or hsa-miR-373 ^*in one or more.Can comprise one or more of miR-148a, miR-122, miR-146a, miR-22 and miR-24 for the biological marking that cancer and non-cancer are distinguished.

The biological marking of the present invention can comprise multiple markers.For example, multiple proteins mark and miR can be used for prostate cancer is distinguished with normal, BPH and PCa, or metastatic disease and non-metastatic disease are distinguished.The sensitivity that can be improved in this way,, specificity and/or accuracy.In some embodiments, one or more the level in hsa-miR-432, hsa-miR-143, hsa-miR-424, hsa-miR-204, hsa-miR-581f and hsa-miR-451 in patient's sample is detected to assess existing of prostate cancer.Any miR in suffering from the patient of PCa in these miR can increase, but the blood-serum P SA of have<4.0ng/ml.In one embodiment, the invention provides the method for assessment prostate cancer, it comprises one or more the level of measuring from hsa-miR-432, hsa-miR-143, hsa-miR-424, hsa-miR-204, hsa-miR-581f and hsa-miR-451 in experimenter's sample.Described sample can be body fluid, for example blood, blood plasma or serum.The described miR of separation in the vesica of described sample can separated.Described experimenter can have the PSA level lower than certain threshold value in blood sample, such as 2.0,2.2,2.4,2.6,2.8,3.0,3.2,3.4,3.6,3.8,4.0,4.2,4.4,4.6,4.8,5.0,5.2,5.4,5.6,5.8 or 6.0ng/ml.Can mean the existence of PCa in described sample higher than the miR level of reference sample.In some embodiments, described reference comprises the level from one or more miR in the experimenter's who does not suffer from PCa control sample.In some embodiments, described reference comprise from patient PCa and PSA level >=certain threshold value (such as 2.0,2.2,2.4,2.6,2.8,3.0,3.2,3.4,3.6,3.8,4.0,4.2,4.4,4.6,4.8,5.0,5.2,5.4,5.6,5.8 or 6.0ng/ml) experimenter's control sample in the level of one or more miR.Described threshold value can be 4.0ng/ml.

The invention provides the assessment to the prostate gland imbalance, comprise the existence or the level that detect one or more circulating biological marks that are selected from the biomarker of above enumerating.Described one or more circulating biological marks also can be selected from BCMA, CEACAM-1, HVEM, IL-1R4, IL-10Rb, Trappin-2, p53, hsa-miR-103, hsa-miR-106b, hsa-miR-10b, hsa-miR-125b, hsa-miR-142-3p, hsa-miR-142-5p, hsa-miR-145, hsa-miR-151-5p, hsa-miR-152, hsa-miR-15a, hsa-miR-181a, hsa-miR-1979, hsa-miR-199a-3p, hsa-miR-19a, hsa-miR-200a, hsa-miR-20b, hsa-miR-29a, hsa-miR-29b, hsa-miR-30a, hsa-miR-329, hsa-miR-335, hsa-miR-361-3p, hsa-miR-365, hsa-miR-373, hsa-miR-423-5p, hsa-miR-502-5p, hsa-miR-595, hsa-miR-663, hsa-miR-671-5p, hsa-miR-760, hsa-miR-7a, hsa-miR-7c, hsa-miR-888, hsa-miR-99a and combination thereof.Described one or more circulating biological marks can be selected from following: hsa-miR-100, hsa-miR-1236, hsa-miR-1296, hsa-miR-141, hsa-miR-146b-5p, hsa-miR-17 ^*, hsa-miR-181a, hsa-miR-200b, hsa-miR-20a ^*, hsa-miR-23a ^*, hsa-miR-331-3p, hsa-miR-375, hsa-miR-452, hsa-miR-572, hsa-miR-574-3p, hsa-miR-577, hsa-miR-582-3p, hsa-miR-937, miR-10a, miR-134, miR-141, miR-200b, miR-30a, miR-32, miR-375, miR-495, miR-564, miR-570, miR-574-3p, miR-885-3p and combination thereof.Further, described one or more circulating biological marks can be selected from following: hsa-let-7b, hsa-miR-107, hsa-miR-1205, hsa-miR-1270, hsa-miR-130b, hsa-miR-141, hsa-miR-143, hsa-miR-148b ^*, hsa-miR-150, hsa-miR-154 ^*, hsa-miR-181a ^*, hsa-miR-181a-2 ^*, hsa-miR-18a ^*, hsa-miR-19b-1 ^*, hsa-miR-204, hsa-miR-2110, hsa-miR-215, hsa-miR-217, hsa-miR-219-2-3p, hsa-miR-23b ^*, hsa-miR-299-5p, hsa-miR-301a, hsa-miR-301a, hsa-miR-326, hsa-miR-331-3p, hsa-miR-365 ^*, hsa-miR-373 ^*, hsa-miR-424, hsa-miR-424 ^*, hsa-miR-432, hsa-miR-450a, hsa-miR-451, hsa-miR-484, hsa-miR-497, hsa-miR-517 ^*, hsa-miR-517a, hsa-miR-518f, hsa-miR-574-3p, hsa-miR-595, hsa-miR-617, hsa-miR-625 ^*, hsa-miR-628-5p, hsa-miR-629, hsa-miR-634, hsa-miR-769-5p, hsa-miR-93, hsa-miR-96.Described circulating biological mark can be one or more in hsa-miR-1974, hsa-miR-27b, hsa-miR-103, hsa-miR-146a, hsa-miR-22, hsa-miR-382, hsa-miR-23a, hsa-miR-376c, hsa-miR-335, hsa-miR-142-5p, hsa-miR-221, hsa-miR-142-3p, hsa-miR-151-3p, hsa-miR-21 and hsa-miR-16.In one embodiment, described circulating biological mark comprises one or more in CD9, PSMA, PCSA, CD63, CD81, B7H3, IL 6, OPG-13, IL6R, PA2G4, EZH2, RUNX2, SERPINB3 and EpCam.Described biomarker can comprise one or more in FOX01A, SOX9, CLNS 1A, PTGDS, XPO1, LETMD1, RAD23B, ABCC3, APC, CHES1, EDNRA, FRZB, HSPG2 and TMPRSS2ETV1 syzygy.Referring to WO2010056993, this application is incorporated to this paper in full by reference.In another embodiment, described circulating biological mark comprises A33, a33n15, AFP, ALA, ALIX, ALP, annexin V, APC, ASCA, ASPH (246-260), ASPH (666-680), ASPH (A-10), ASPH (D01P), ASPH (D03), ASPH (G-20), ASPH (H-300), AURKA, AURKB, B7H3, B7H4, BCA-225, BCNP1, BDNF, BRCA, CA125 (MUC16), CA-19-9, C-Bir, CD1.1, CD10, CD174 (Lewis y), CD24, CD44, CD46, CD59 (MEM-43), CD63, CD66e CEA, CD73, CD81, CD9, CDA, CDAC11a2, CEA, C-Erb2, C-erbB2, CRMP-2, CRP, CXCL12, CYFRA21-1, DLL4, DR3, EGFR, Epcam, EphA2, EphA2 (H-77), ER, ErbB4, EZH2, FASL, FRT, FRT c.f23, GDF15, GPCR, GPR30, Gro-α, HAP, HBD 1, HBD2, HER 3 (ErbB3), HSP, HSP70, hVEGFR2, iC3b, IL 6Unc, IL-1B, IL6 Unc, IL6R, IL8, IL-8, INSIG-2, KLK2, L1CAM, LAMN, LDH, MACC-1, MAPK4, MART-1, MCP-1, M-CSF, MFG-E8, MIC1, MIF, MIS RII, MMG, MMP26, MMP7, MMP9, MS4A1, MUC1, MUC1 seq1, MUC1 seq11A, MUC17, MUC2, Ncam, NGAL, NPGP/NPFF2, OPG, OPN, p53, p53, PA2G4, PBP, PCSA, PDGFRB, PGP9.5, PIM1, PR (B), PRL, PSA, PSMA, PSME3, PTEN, R5-CD9 Tube 1, Reg IV, RUNX2, SCRN1, seprase, SERPINB3, SPARC, SPB, SPDEF, SRVN, STAT 3, STEAP1, TF (FL-295), TFF3, TGM2, TIMP-1, TIMP1, TIMP2, TMEM211, TMPRSS2, TNF-α, Trail-R2, Trail-R4, TrKB, TROP2, Tsg 101, TWEAK, UNC93A, one or more in VEGF A and YPSMA-1.In the biological marking, can use the arbitrary combination of these marks with the assessment prostate cancer.Described circulating biological mark can be associated with vesica, as vesica surface marker or vesica useful load.The imbalance of described prostate cancer includes but not limited to optimum obstacle (such as BPH) or prostate cancer, comprise various differences by stages with the cancer of grade.For example,, referring to table 5:

Table 5: for the biomarker of prostate gland imbalance

The arbitrary combination of these marks can be used in the biological marking with the imbalance of assessment prostate gland (such as BPH) and prostate cancer.The described biological marking also can be used for assessing described prostate cancer by stages or grade.

Can characterize prostate cancer with at least 60,61,62,63,64,65,66,67,68,69 or 70% sensitivity by one or more methods disclosed herein.Can characterize prostate cancer with at least 80,81,82,83,84,85,86 or 87% sensitivity.For example, can be with at least 87.1,87.2,87.3,87.4,87.5,87.6,87.7,87.8,87.9,88.0 or 89% sensitivity (for example, with at least 90% sensitivity, for example at least 91,92,93,94,95,96,97,98,99 or 100% sensitivity) characterize prostate cancer.

Can also be with at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96 or 97% specificity is (for example, with at least 97.1, 97.2, 97.3, 97.4, 97.5, 97.6, 97.7, 97.8, 97.8, 97.9, 98.0, 98.1, 98.2, 98.3, 98.4, 98.5, 98.6, 98.7, 98.8, 98.9, 99.0, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 100% specificity) characterize experimenter's prostate cancer.

In addition, can also be with at least 70% sensitivity and at least 80,90,95,99 or 100% specificity; At least 80% sensitivity and at least 80,85,90,95,99 or 100% specificity; At least 85% sensitivity and at least 80,85,90,95,99 or 100% specificity; At least 86% sensitivity and at least 80,85,90,95,99 or 100% specificity; At least 87% sensitivity and at least 80,85,90,95,99 or 100% specificity; At least 88% sensitivity and at least 80,85,90,95,99 or 100% specificity; At least 89% sensitivity and at least 80,85,90,95,99 or 100% specificity; At least 90% sensitivity and at least 80,85,90,95,99 or 100% specificity; At least 95% sensitivity and at least 80,85,90,95,99 or 100% specificity; At least 99% sensitivity and at least 80,85,90,95,99 or 100% specificity; Perhaps at least 100% sensitivity and at least 80,85,90,95,99 or 100% specificity characterize prostate cancer.

In some embodiments, the described biological marking is with at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96 or 97% accuracy is (for example, with at least 97.1, 97.2, 97.3, 97.4, 97.5, 97.6, 97.7, 97.8, 97.8, 97.9, 98.0, 98.1, 98.2, 98.3, 98.4, 98.5, 98.6, 98.7, 98.8, 98.9, 99.0, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 100% accuracy) characterize experimenter's phenotype.

In some embodiments, the described biological marking is with at least 0.70, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96 or 0.97 AUC is (for example, with at least 0.971, 0.972, 0.973, 0.974, 0.975, 0.976, 0.977, 0.978, 0.978, 0.979, 0.980, 0.981, 0.982, 0.983, 0.984, 0.985, 0.986, 0.987, 0.988, 0.989, 0.99, 0.991, 0.992, 0.993, 0.994, 0.995, 0.996, 0.997, 0.998, 0.999 or 1.00 AUC) characterize experimenter's phenotype.

In addition, can be identified for measuring with at least 70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98 or 99% degree of confidence the confidence level of specificity, sensitivity, accuracy and/or AUC.

Gastric and intestinal cancer

Described stomach and intestine (GI) road includes but not limited to that oral cavity, gum, gum, tongue, sialisterium, oesophagus, pancreas, liver, gall-bladder, small intestine (duodenum, jejunum, ileum), bile duct, stomach are dirty, large intestine (caecum, colon, rectum), appendix and anus.The biological marking of described biomarker can be used for detecting or characterizing the cancer of these components, as colorectal carcinoma (CRC), cancer of the stomach, intestinal cancer, liver cancer or the esophageal carcinoma.The biological marking in stomach and intestine (GI) road can comprise listed any one in Fig. 1 or plurality of antigens, for example, for (characterizing any one relevant with vesica that separate colorectal carcinoma or multiple wedding agent, as shown in Figure 2), any one or multiple other biomarker, as shown in Figure 6.

Colorectal carcinoma

Although colonoscopy is examination and the gold standard of confirming colorectal carcinoma (CRC), half is proposed the patient who carries out colonoscopy and does not comply with according to estimates.The shortage of compliance is normally because a lot of people feel that colonoscopy is discomfort and the invasive process of tool.The low invasive diagnostic test that can differentiate the patient of the biological marking (its indication detects and bioptic needs by colonoscopy) had based on blood can improve compliance.This strategy will make cancer more early be identified and prevent to be subject to unnecessary invasive procedures without the individuality of disease.The current test based on blood depends on the level increased of carcinomebryonic antigen (CEA) or Carbohydrate Antigens determinant (CA 19-9).Unfortunately, CEA and CA 19-9 are neither organ specific also non-tumour-specific.The present invention improves the detection analysis based on vesica of using these marks.

The invention provides and used the biological marking be derived from from experimenter's sample to identify the method for likely suffering from or just suffering from the experimenter of CRC.Described sample can be body fluid (such as blood, blood plasma or serum) or ight soil.The described biological marking can comprise the circulating biological mark, comprises the biomarker relevant to vesica.The described biological marking can comprise the sudden change by nucleic acid (as the RNA comprised in vesica) is checked order and detects.

In one aspect of the invention, the biological marking is derived from the vesica separated from suffering from and do not suffer from patient's blood plasma of CRC.The vesica surface protein is used for multiple analysis to catch and to detect vesica.The amount of vesica with these surface proteins of remarkable concentration causes forming can distinguish CRC sample and the normal vesica specific biological marking.These vesicas that exist in CRC patient's blood plasma provide can be in the situation that early to 1 grade of marking of diagnosing CRC of histology.In some embodiments, use the antibody capture vesica for various surface proteins.The vesica of catching can be used the vesica Specific marker to be detected, as one or more in CD9, CD81 and CD63.In some embodiments, the biological marking based on vesica comprises one or more the level of measuring in CD9, CD81, CD63, EpCam, EGFR and STEAP.In some embodiments, one or more following marks are used for catching and/or detecting vesica: CD9, NGAL, CD81, STEAP, CD24, A33, CD66E, EPHA2, TMEM211, TROP2, TROP2, EGFR, DR3, UNC93A, MUC17, EpCAM, MUC17, CD63, B7H3.In some embodiments, one or more following marks are used for catching and/or detecting vesica: TMEM211, MUC1, GPR110 (GPCR 110), CD24, CD9, CD81 and CD63.In some embodiments, one or more following marks are used for catching and/or detecting vesica: DR3, STEAP, epha2, TMEM211, unc93A, A33, CD24, NGAL, EpCam, MUC17, TROP2 and TETS.In some embodiments, TMEM211 is for catching and/or detect vesica.In some embodiments, MUC1 is for catching and/or detect vesica.In some embodiments, GPR110 is for catching and/or detect vesica.In some embodiments, CD24 is for catching and/or detect vesica.In some embodiments, by one or more in TMEM211, MUC1, GPR110 (GPCR 110) and CD24 for catching vesica and one or more general vesica marks for detection of described captive vesica.

In another aspect of this invention, the microRNA relevant to vesica (miR) is for determining the biological marking.Described miR can be derived from the vesica (as exosome) separated from patient's sample (as blood).In some embodiments, one or more following miR are for generation of the CRC biological marking: miR 92, miR 21, miR 9 and miR 491.

Again aspect another, assessed the useful load in vesica of the present invention.KRAS and BRAF Mutation Screening can be used for the colorectal carcinoma monitoring to tumor sample.As described in Example 4, the KRAS sudden change sees the RNA that is derived from the colon cell line vesica.Embodiment 5 shows in being derived from the RNA vesica of plasma sample, to KRAS, to be checked order.In some embodiments, the order-checking of the KRAS in vesica and/or BRAF nucleic acid be can be used for detecting CRC.Described nucleic acid can be RNA, as mRNA.The biological marking of CRC can comprise separating the order-checking from KRAS and the BRAF RNA of vesica.

The biological marking of colorectal carcinoma can comprise as listed any one or plurality of antigens for colorectal carcinoma in Fig. 1, any one or multiple with separate or detect the relevant wedding agent (for example, as shown in Fig. 2) of vesica for characterizing colorectal carcinoma, any one or multiple other biomarker.As shown in Figure 6.

The described biological marking can comprise that one or more are selected from miR-24-1, miR-29b-2, miR-20a, miR-10a, miR-32, miR-203, miR-106a, miR-17-5p, miR-30c, miR-223, miR-126, miR-128b, miR-21, miR-24-2, miR-99b, miR-155, miR-213, miR-150, miR-107, miR-191, miR-221, miR-20a, miR-510, miR-92, miR-513, miR-19a, miR-21, miR-20, miR-183, miR-96, miR-135b, miR-31, miR-21, miR-92, miR-222, miR-181b, miR-210, miR-20a, miR-106a, miR-93, miR-335, miR-338, miR-133b, miR-346, miR-106b, miR-153a, miR-219, miR-34a, miR-99b, miR-185, miR-223, miR-211, miR-135a, miR-127, miR-203, miR-212, miRNA in miR-95 or miR-17-5p or their any combination.The described biological marking can also comprise that one or more express not enough miR, miR-143 for example, miR-145, miR-143, miR-126, miR-34b, miR-34c, let-7, miR-9-3, miR-34a, miR-145, miR-455, miR-484, miR-101, miR-145, miR-133b, miR-129, miR-124a, miR-30-3p, miR-328, miR-106a, miR-17-5p, miR-342, miR-192, miR-1, miR-34b, miR-215, miR-192, miR-301, miR-324-5p, miR-30a-3p, miR-34c, miR-331, miR-148b, miR-548c-5p, miR-362-3p and miR422a.

The described biological marking can comprise assessment one or more genes, for example EFNB1, ERCC1, HER2, VEGF and EGFR.The biomarker sudden change of the colorectal carcinoma that can assess in vesica in addition, can also comprise one or more sudden changes of EGFR, KRAS, VEGFA, B-Raf, APC or p53.The described biological marking can also comprise one or more protein, part or the peptide of appreciable vesica, for example AFR, Rab, ADAM10, CD44, NG2, Ephrin-B1, MIF, b-catenin, joint, plakoglobin, half lactadherin-4, RACK1, four transmembrane proteins-8, FasL, TRAIL, A33, CEA, EGFR, pepx 1, hsc-70, four transmembrane proteins, ESCRT, TS, PTEN or TOPO1.

Can separate and analyze vesica to compose for providing diagnosis, prognosis or treatment to diagnose, for example the further feature of the effect of the stage of cancer, cancer or cancer.Perhaps, can be by sample direct analysis vesica, thus for before being analyzed with the Cancer-Related biological marking of colon, described vesica is not purified or concentrated.

As shown in Figure 69, for the GI cancer such such as colorectal carcinoma, the biological marking can comprise EpCam, CD63, CD81, CD9, CD66 or their any combination that detects vesica.In addition, can comprise CD63, CD9, EpCam or their any combination (for example, referring to Figure 71 and Figure 72) for the biological marking of the colorectal carcinoma of each different steps of cancer.For example, the described biological marking can comprise CD9 and EpCam.In some embodiments, the biological marking of described GI cancer comprises one or more miRNA that are selected from miR-548c-5p, miR-362-3p, miR-422a, miR-597, miR-429, miR-200a and miR-200b.As shown in Figure 110, these miRNA can cross and express in the GI cancer.The described miRNA marking can combine with above listed biomarker.The described biological marking can provide diagnosis, prognosis or treatment diagnosis spectrum, for example further feature of the effect of the stage of cancer, cancer or cancer.

The invention provides the assessment to gastrointestinal disorder, it comprises existence and the level that detects one or more circulating biological marks that are selected from the biomarker of above enumerating.Described one or more circulating biological marks can be selected from CD9, EGFR, NGAL, CD81, STEAP, CD24, A33, CD66E, EPHA2, ferritin, GPR30, GPR110, MMP9, OPN, p53, TMEM211, TROP2, TGM2, TIMP, EGFR, DR3, UNC93A, MUC17, EpCAM, MUC1, MUC2, TSG101, CD63, B7H3 and combination thereof.Described one or more circulating biological marks can be selected from following: DR3, STEAP, epha2, TMEM211, unc93A, A33, CD24, NGAL, EpCam, MUC17, TROP2, TETS and combination thereof.Further again, described one or more circulating biological marks are selected from following: A33, AFP, ALIX, ALX4, ANCA, APC, ASCA, AURKA, AURKB, B7H3, BANK1, BCNP1, BDNF, CA-19-9, CCSA-2, CCSA-3& 4, CD10, CD24, CD44, CD63, CD66CEA, CD66eCEA, CD81, CD9, CDA, C-Erb2, CRMP-2, CRP, CRTN, CXCL12, CYFRA21-1, DcR3, DLL4, DR3, EGFR, Epcam, EphA2, FASL, FRT, GAL3, GDF15, GPCR (GPR110), GPR30, GRO-1, HBD 1, HBD2, HNP1-3, IL-1B, IL8, IMP3, L1CAM, LAMN, MACC-1, MGC20553, MCP-1, M-CSF, MIC1, MIF, MMP7, MMP9, MS4A1, MUC1, MUC17, MUC2, Ncam, NGAL, NNMT, OPN, p53, PCSA, PDGFRB, PRL, PSMA, PSME3, Reg IV, SCRN1, Sept-9, SPARC, SPON2, SPR, SRVN, TFF3, TGM2, TIMP-1, TMEM211, TNF-α, TPA, TPS, Trail-R2, Trail-R4, TrKB, TROP2, Tsg 101, TWEAK, UNC93A and VEGFA and combination thereof.In one embodiment, described circulating biological mark comprises one or more in one or more and/or hsa-miR-376c, hsa-miR-215, hsa-miR-652, hsa-miR-582-5p, hsa-miR-324-5p, hsa-miR-1296, hsa-miR-28-5p, hsa-miR-190, hsa-miR-590-5p, hsa-miR-202 and the hsa-miR-195 in miR 92, miR 21, miR 9 and miR 491.In another embodiment, described circulating biological mark comprises one or more in TMEM211, MUC1, CD24 and/or GPR110 (GPCR 110).Described circulating biological mark can be associated with vesica, as vesica surface marker or vesica useful load.Described gastrointestinal disorder includes but not limited to optimum imbalance (such as benign polypus) or cancer (such as colorectal carcinoma), it comprise various by stages with the cancer of grade.For example,, referring to table 6.

Table 6: for the biomarker of gastrointestinal disorder

Can characterize colorectal carcinoma by one or more methods as herein described with at least 60,61,62,63,64,65,66,67,68,69 or 70% sensitivity.Can characterize colorectal carcinoma with at least 71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86 or 87% sensitivity.For example, can characterize colorectal carcinoma with at least 87.1,87.2,87.3,87.4,87.5,87.6,87.7,87.8,87.9,88.0 or 89% sensitivity (for example, for example, with at least 90% sensitivity, at least 91,92,93,94,95,96,97,98,99 or 100% sensitivity).

Can also be with at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96 or 97% specificity is (for example, with at least 97.1, 97.2, 97.3, 97.4, 97.5, 97.6, 97.7, 97.8, 97.8, 97.9, 98.0, 98.1, 98.2, 98.3, 98.4, 98.5, 98.6, 98.7, 98.8, 98.9, 99.0, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 100% specificity) characterize experimenter's colorectal carcinoma.

Can also be with at least 70% sensitivity and at least 80,90,95,99 or 100% specificity; At least 80% sensitivity and at least 80,85,90,95,99 or 100% specificity; At least 85% sensitivity and at least 80,85,90,95,99 or 100% specificity; At least 86% sensitivity and at least 80,85,90,95,99 or 100% specificity; At least 87% sensitivity and at least 80,85,90,95,99 or 100% specificity; At least 88% sensitivity and at least 80,85,90,95,99 or 100% specificity; At least 89% sensitivity and at least 80,85,90,95,99 or 100% specificity; At least 90% sensitivity and at least 80,85,90,95,99 or 100% specificity; At least 95% sensitivity and at least 80,85,90,95,99 or 100% specificity; At least 99% sensitivity and at least 80,85,90,95,99 or 100% specificity; Perhaps at least 100% sensitivity and at least 80,85,90,95,99 or 100% specificity characterize colorectal carcinoma.

In addition, can there is at least 70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98 or 99% degree of confidence for measuring the confidence level that specificity, sensitivity, accuracy and/or other statistic property measure.

Ovarian cancer

For the biological marking that characterizes ovarian cancer, can comprise and the Cancer-Related antigen of ovary (for example, shown in Fig. 1), and one or more other biomarkers, for example, shown in Fig. 4.In one embodiment, the biological marking of ovarian cancer can comprise one or more antigen relevant to ovarian cancer, such as, but not limited to, CD24, CA125, VEGF1, VEGFR2, HER2, MISIIR or their any combination.The biological marking of ovarian cancer can comprise one or more aforesaid antigens and one or more other biomarkers, such as but not limited to miRNA, mRNA, DNA or their any combination.The biological marking of ovarian cancer can comprise one or more antigen relevant to ovarian cancer, such as, but not limited to CD24, CA125, VEGF1, VEGFR2, HER2, MISIIR, or their any combination, one or more miRNA biomarkers, such as, but not limited to, miR-200a, miR-141, miR-200c, miR-200b, miR-21, miR-141, miR-200a, miR-200b, miR-200c, miR-203, miR-205, miR-214, miR-215, miR-199a, miR-140, miR-145, miR-125b-1 or their any combination.

The biological marking of ovarian cancer can comprise one or more antigen relevant to ovarian cancer, such as, but not limited to CD24, CA125, VEGF1, VEGFR2, HER2, MISIIR or their any combination, and one or more miRNA biomarkers (such as aforementioned miRNA), mRNA (such as, but not limited to ERCC1, ER, TOPO1, TOP2A, AR, PTEN, HER2/neu, EGFR), sudden change (including but not limited to the sudden change relevant to KRAS and/or B-Raf) or their any combination.

Can for one or more miRNA with separate, analyze or separate and analyze vesica with Cancer-Related one or more antigens of ovary, thereby diagnosis, prognosis or treatment diagnosis spectrum are provided.Perhaps, can be by sample direct analysis vesica, before the miRNA relevant to ovarian cancer for one or more or antigen are analyzed, described vesica is not purified or concentrated thus.

Organ-graft refection and auto immune conditions

Vesica can also for example, for determining phenotype, organ desperate situation and/or organ-graft refection.As used herein, organ transplantation comprises the transplanting of part organ or tissue.Can assess the existence of one or more biomarkers that exist in vesica, do not exist or level with monitoring organ rejection or survive.In sample, the level of vesica or amount can also or survive for assessment of the organ rejection.In addition, can determine the assessment situation with at least 70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98 or 99% specificity, sensitivity or the two.For example, can determine the assessment situation with at least 97.5,97.6,97.7,97.8,97.8,97.9,98.0,98.1,98.2,98.3,98.4,98.5,98.6,98.7,98.8,98.9,99.0,99.1,998.2,99.3,99.4,99.5,99.6,99.7,99.8,99.9% sensitivity, specificity or these two.

Can be before analyzing purifying or concentrating vesicles.Perhaps, can be in the situation that without purifying in advance or concentrated directly from level or the amount of sample analysis vesica.Described vesica can be by quantitatively.For example, can come isolated cell or tissue-specific vesica with certain organs being there are to specific one or more wedding agents.Can for example, for one or more characterization of molecules (one or more biomarkers relevant with organ desperate situation or organ-graft refection), assess cell source specificity vesica.Can assess the existence of existing one or more biomarkers, do not exist or level with monitoring organ rejection or survive.

Can analyze one or more vesicas with for assessment of, detect or diagnosis experimenter's tissue or the repulsion of organ transplantation.Described tissue or organ-graft refection can be super acute, acute or chronic rejection.In addition, can also analyze vesica with for assessment of, detect or diagnosis experimenter's graft versus host disease (GVH disease).Described experimenter can be autologous, allogeneic or the tissue of xenogenesis or the recipient of organ transplantation.

Can also analyze vesica to detect the repulsion of tissue or organ transplantation.Described vesica can produce by tissue or organ transplantation.These tissues or organ include but not limited to the cell of heart, lung, pancreas, kidney, eyes, cornea, muscle, marrow, skin, cartilage, bone, accessory organ, hair, face, tendon, stomach, intestines, vein, artery, differentiation, cell or the stem cell of part differentiation.

Described vesica can comprise at least one biomarker, and described biomarker is possibility or the generation to the repulsion of tissue or organ transplantation for assessment of, diagnosis or definite experimenter.Biomarker can also or detect experimenter's graft versus host disease (GVH disease) for assessment of, diagnosis.Described biomarker can be protein, polysaccharide, lipid acid or nucleic acid (for example DNA or RNA).Described biomarker can be relevant with specific tissue or organ rejection or systemic organ failure.Can analyze the biomarker more than, for example, one or more protein markers can with one or more nucleic acid mark binding analysis.Described biomarker can be in cell or extracellular mark.

Can analyze described vesica at least one mark, with for assessment of, detect or diagnosis is relevant to or causes tissue in the experimenter or organ-graft refection's apoptosis or necrosis.

The existence of biomarker can be the indication of tissue or organ rejection in the experimenter, wherein said biomarker includes but not limited to CD40, CD40L, N-acetyl muramyl-ALANINE Ntn hydrolase precursor, fat connection albumen, the AMBP amyloid protein precursor, C4b-conjugated protein a chain precursor, the ceruloplasmin precursor, the complement Complement C3 precursor, the complement component C9 precursor, the Complement Factor D precursor, α 1-B-glycoprotein, beta 2-glycoprotein I precursor, heparin cofactor II precursor, immunoglobulin mu chain C district albumen, be rich in leucic α 2-glycoprotein precursor, pigment epithelial cell source factor precursor, blood plasma retinol conjugated protein precursor, translation initiation factor 3 subunit 10s, ribosome protein L 7/L, β-transducer, 1-TRAF or lysyl-tRNA synthetic enzyme.

Thereby also can analyze the kidney repulsion that vesica detects the experimenter by the existence for β-transducer.Can also by the specific vesica in isolated cell source the cell from expressing CD40 and detect Bcl-2 or the increase of TNF α to detect the repulsion of transplanted tissue.

Thereby can analyze vesica by the existence for F1 antigen mark and detect the repulsion of experimenter to liver transplantation.Be not limited by theory, having can be for detection of the increase of the specific vesica in liver cell source to the specific F1 antigen of liver.This increase can be as the early stage index of organ desperate situation/repulsion.

In addition, can also diagnose the bronchiolitis obliterans or the transplanted abdominal sclerosis/transplanted veins that cause due to marrow and/or lung transplantation or other reason to harden by analyzing vesica.

In addition, can also analyze vesica with for detection of, diagnosis or autoimmunization or other the immune response relevant phenotype of assessment in the experimenter.The example of this imbalance includes but not limited to systemic lupus erythematous (SLE), discoid lupus, systemic lupus erythematosus, sarcoidosis, inflammatory arthritis (comprises sacroiliitis childhood, rheumatoid arthritis, psoriatic arthritis, Reiter syndrome, ankylosing spondylitis and urarthritis), multiple sclerosis, hyper-IgE syndrome, polyarteritis nodosa, primary biliary cirrhosis, inflammatory bowel, Crohn's disease, celiac disease (gluten susceptibility enteropathy), autoimmune hepatitis, pernicious anemia, autoimmune hemolytic anemia, psoriatic, scleroderma, myasthenia gravis, autoimmune thrombocytopenic purpura, autoimmune thyroiditis, Graves disease, Hashimoto thyroiditis, the immunocomplex disease, confirmed fatigue immunologic function disorder syndrome (CFIDS), PM-DM, cryoglobulinemia, thrombolysis, myocardosis, pemphigus vulgaris, interstitial pulmonary fibrosis, asthma, Churg-Strauss syndrome (allergic granuloma), atopic dermatitis, allergy and irritant contact dermatitis, urticaria, the allergy of IgE mediation, atherosclerosis, vasculitis, the idiopathic inflammatory myopathy, Hemolysis, alzheimer's disease, chronic inflammatory demyelinating polyneuropathy and AIDS.

The possibility that can occur for assessment of the autoimmunization in, diagnosis or definite experimenter or the imbalance of other immune response dependency from one or more biomarkers of vesica.Described biomarker can be protein, polysaccharide, lipid acid or nucleic acid (for example DNA or RNA).Described biomarker can be lacked of proper care relevant with specific Autoimmune Disorders, systemic Autoimmune Disorders and other immune response dependency.Can analyze more than a kind of biomarker.For example, one or more protein markers can with one or more nucleic acid mark combinatory analyses.Described biomarker can be in cell or extracellular mark.In addition, described biomarker can for detection of, the diagnosis or the assessment inflammation.

Analysis to the vesica from the experimenter can be used for identifying the experimenter who suffers from the inflammation for example, with anaphylactic disease (pulmonary fibrosis that hyperergy pneumonia, eosinophilic granulocyte pneumonia and collagen protein cause), the vasculitis of asthma, osteitis tuberculosa cystica, pulmonary emphysema, cystic fibrosis, idiopathic pulmonary fibrosis, chronic bronchitis, allergic rhinitis and lung for example, with autoimmune disease (rheumatoid arthritis) relevant.

The treatment diagnosis

According to disclosed herein, disclose for characterize the method for experimenter's phenotype by one or more biomarkers of assessment (comprising vesica biomarker and/or circulating biological mark).Described biomarker can be used the method that vesica biomarker disclosed herein is carried out to multiple analysis to be assessed.Characterize phenotype the treatment diagnosis provided the experimenter can be provided, such as whether definite experimenter is predicted, treatment is had to reaction or predicted reactionless to treating.But experimenter's called after respondent that treatment is responded, but and the non-respondent of unresponsive experimenter's called after.According to but be not limited to the improvement of one or more symptoms of situation; The reduction of one or more side effects of existing treatment; With before this or other treatment the compare improvement of one or more symptoms or the raising of improvement rate; With treated or with before this or other treatment compare longer survival time, can think that the experimenter who suffers from described situation is the respondent of described treatment.For example, can think that according to useful or required treatment consequence the experimenter of the situation of suffering from is the respondent of described treatment, described treatment consequence includes but not limited to, the reduction of the improvement of one or more symptoms or alleviation, disease degree, the stabilization of morbid state are (, have no deterioration), the delay of prophylactic diffusion, disease process or slow down, the alleviation of morbid state or alleviate and no matter partially or completely the course of disease alleviates (), be no matter can detect or non-detectable.The treatment also comprise from the situation that do not receive treatment or accept the survival time that the desired survival time of different treatments is compared prolongation.

The patient that system and method disclosed herein can be used for for demand is arranged selects candidate therapeutic.One or more features that can be based on vesica to the selection of therapy, such as the biological marking of vesica, vesica amount or the two.The typing of vesica or somatotype (such as the identity of the biological marking of vesica, vesica amount or the two) can be used for one or more candidate therapeutic agents of Individual identification for suffering from situation.For example, the vesica somatotype can be used for determining that the experimenter is non-respondent or respondent for specific therapy (such as described experimenter, suffering from the cancer therapy in cancered situation).

The vesica somatotype can be used for providing treatment or prognosis to the experimenter, and can select therapy according to described diagnosis or prognosis.Perhaps, the directly spectrum of the vesica based on the experimenter is selected in treatment.In addition, experimenter's vesica spectrum can be used for following the trail of the evolution of disease, for assessment of pharmaceutical efficacy, the experimenter that existing treatment adapted to suffer from disease or situation or select new treatment for the experimenter who suffers from disease or situation.

The experimenter can use biomarker to be assessed to the reaction for the treatment of, and it comprises vesica, microRNA and other circulating biological mark.In one embodiment, the spectrum of the experimenter's vesica based on carrying out assessing before any treatment is determined described experimenter, classify or is accredited as non-respondent or respondent.During pretreat, the experimenter can be categorized as to non-respondent or respondent, reduce therefrom unnecessary treatment option, and avoided the side effect from invalid therapy.In addition, described experimenter can be accredited as to the respondent for particular treatment, and the survival time by providing the personalized treatment option to extend the experimenter is provided the vesica somatotype thus, improves described experimenter's symptom or situation or the two.Therefore, the experimenter who suffers from situation can have the biological marking that uses one or more system and methods disclosed herein to be produced by vesica and other circulating biological mark, and described spectrum can be subsequently for determining that the experimenter is non-respondent or respondent for the particular treatment possibility of described situation.According to for predicting whether the treatment that described experimenter is used for the treatment of described experimenter's situation for initial imagination is the use of non-respondent or respondent's biomarker, can be described experimenter and select imagination to be used for the treatment of described experimenter's the particular treatment of situation, maybe can select the treatment that other may be better.

In some embodiments, the experimenter who suffers from situation just is being subject to the treatment of therapeutical agent.Can be before treatment and treatment during one or more time points obtain sample from described experimenter.Can assess and comprise from the biological marking of the vesica of described sample or other biomarker and use it for and determine the reaction of described experimenter for described medicine, such as according to the described biological marking over time.If described experimenter is reactionless for described treatment, for example, the described biological marking does not show that described patient reacts, and described experimenter can be classified as for described treatment anergy, or is non-respondent.Similarly, thus can detect one or more biomarkers relevant to the situation worsened makes described biological marking indication patient to described treatment, not produce favourable reaction.In another embodiment, although treat, one or more biomarkers relevant to described situation remain unchanged, and this has shown that described situation do not improve.Therefore, according to the described biological marking, can change or adjust the treatment plan for described experimenter, comprise and select different therapeutical agents.

Perhaps, can determine that described experimenter responds for described treatment, and described experimenter can be categorized as for described treatment and have reactivity, or be the respondent.For example, can detect one or more biomarkers relevant to the improvement of described situation or imbalance.In another example, one or more biomarkers relevant to described situation change to some extent, thereby have shown improvement.Therefore, existing treatment can continue.In another embodiment, though the sign exist improved, the described biological marking shown another kind for the treatment of may more efficiently situation under capable of regulating or change existing treatment.Existing treatment can increase the dosage of current therapeutical agent in conjunction with another therapeutical agent, or selects different candidate therapeutic or therapeutical agent.For selecting the standard of different candidate therapeutic to can be depending on setting.In one embodiment, described candidate therapeutic possibility is known is effective for the successful experimenter of existing treatment.In another embodiment, described candidate therapeutic may known other experimenter for having the similar biological marking be effective.

In some embodiments, described experimenter is being subject to second, third or more multiclass treatment, such as cancer therapy.Can carry out second, third or more before multiclass treatment, described experimenter determined to the biological marking of the present invention, take determine the experimenter for described second, third or more the multiclass treatment whether be respondent or non-respondent.In another embodiment, carrying out second, third or more during multiclass treatment, described experimenter determined to the biological marking, thus determine the experimenter for described second, third or more the multiclass treatment whether respond.

Method and system for assessment of one or more vesicas as herein described can be used for determining whether the experimenter of the situation of suffering from has reactivity for treatment, and therefore can be used for the treatment that selection improves one or more symptoms of described situation; Reduce one or more side effects of existing treatment; With before this or other treatment compare and improve improvement or its improvement speed of one or more symptoms; With treated or with before this or other treatment compare the prolongation survival time.Therefore, method as herein described can be by personalized treatment option is provided for extending experimenter's survival time, and/or can be the experimenter and reduce unnecessary treatment option and unnecessary side effect.

The survival time of described prolongation can be improve without progression of disease survival rate (PFS), the individuality of suffering from disease (as cancer) that it refers to or colony keep the probability without progression of disease after starting the course for the treatment of.It can refer to after the time durations of appointment in described colony disease may keep stable (as, show the progress sign) the per-cent of individuality.The progresson free survival rate is the indication of particular treatment validity.In other embodiments, the survival rate of described prolongation is without disease survival rate (DFS), and it refers to suffers from cancered individuality or group of individuals keep the probability without disease after starting particular treatment.It can refer to after the time durations of appointment in described colony may be without the per-cent of the individuality of disease.It without the disease survival rate, is the indication of particular treatment validity.Can by similar patient colony, obtain without the basis of disease survival rate on two kinds of therapeutic strategies relatively.When describing the cancer survival, without the disease survival rate, usually together with term overall survival rate, use.

According to the candidate therapeutic of selecting by the vesica somatotype described herein, can compare with the treatment that non-vesica somatotype is selected, on its progresson free survival (PFS) by will use the therapy by vesica somatotype (B phase) selection and time that described experimenter has just occurred to make progress, the PFS (A phase) of the most approaching therapy is compared and is carried out.In a kind of setting, >=1.3 PFSB/PFSA provides benefit (for example than the therapy for showing described vesica somatotype selection for the experimenter, referring to Robert Temple, Clinical measurement in drug evaluation.Wu Ningano and G.T.Thicker write, John Wiley and Sons Ltd.1995; Von Hoff, D.D.Clin Can Res.4:1079,1999; Dhani etc., Clin Cancer Res.15:118-123,2009).

Other method that the treatment that the vesica somatotype is selected compares can and get nowhere or experimenter's per-cent of death and comparing with the selected treatment of non-vesica somatotype by assaying reaction rate (RECIST) in 4 months.The term " about " of using in the situation of the numerical value of PFS refers to the change of relatively described numerical value +/-ten Percent (10%).With the treatment that non-vesica somatotype is selected, compare, the PFS of the treatment of selecting from the vesica somatotype can enlarge at least 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or at least 90%.In some embodiments, with the treatment that non-vesica somatotype is selected, compare, the PFS of the treatment of selecting from the vesica somatotype can enlarge at least 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or at least about 1000%.In other embodiment again, described PFS ratio (PFS of the PFS/ of the therapy that the vesica somatotype is selected or new treatment therapy or treatment before this) is at least about 1.3.In other embodiment again, described PFS ratio is at least about 1.1,1.2,1.3,1.4,1.5,1.6,1.7,1.8,1.9 or 2.0.In other embodiment again, described PFS ratio is at least about 3,4,5,6,7,8,9 or 10.

Similarly, can be in the situation that more described DFS in determining or determine the experimenter that the biological marking according to the present invention selected its treatment.With the treatment that non-vesica somatotype is selected, compare, the DFS of the treatment of selecting from the vesica somatotype can enlarge at least 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or at least 90%.In some embodiments, with the treatment that non-vesica somatotype is selected, compare, the DFS of the treatment of selecting from the vesica somatotype can enlarge at least 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or at least about 1000%.In other embodiment again, described DFS ratio (DFS of the DFS/ of the therapy that the vesica somatotype is selected or new treatment therapy or treatment before this) is at least about 1.3.In other embodiment again, described DFS ratio is at least about 1.1,1.2,1.3,1.4,1.5,1.6,1.7,1.8,1.9 or 2.0.In other embodiment again, described DFS ratio is at least about 3,4,5,6,7,8,9 or 10.

In some embodiments, the candidate therapeutic of selecting by the microcapsule bubble somatotype does not improve described PFS ratio or DFS ratio in the experimenter; Although the vesica somatotype provides benefit to the experimenter.For example, in some embodiments, without known treatment, can be used for described experimenter.In these cases, the vesica somatotype provides in the situation that currently do not identify the method that candidate therapeutic has been identified in treatment.Described vesica somatotype can be by PFS, DFS or at least 1 week of life, 2 weeks, 3 weeks, 4 weeks, 1 month, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 2 months, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months or 2 years.Described vesica somatotype can extend PFS, DFS or life-span at least

year, 3 years, 4 years, 5 years or longer.In some embodiments, thus method of the present invention improved result and make the experimenter in alleviating.

Can measure the usefulness that detects treatment by other.The completely dissolve that complete reaction (CR) has comprised described disease: in inspection, scanning or other test, do not prove disease.Partial reaction (PR) refers to and retains in vivo some disease, but at size of tumor or quantitatively reduced 30% or more.Stable disease (SD) refers at size of tumor and quantitatively keeps geostationary disease.Generally, can be described to stable disease lower than 50% reduction or slight raising in size.PD (PD) refer to when treatment described disease in size or quantitatively increase.In some embodiments, vesica somatotype according to the present invention has caused complete reaction or partial reaction.In some embodiments, method of the present invention has caused stable disease.In some embodiments, the present invention can realize stable disease, but not the vesica somatotype has caused PD.

Can be for phenotype according to the treatment diagnosis of the biological marking of the present invention, it includes but not limited to those phenotypes cited herein.Characterize phenotype and be included as the definite treatment diagnosis of experimenter, such as prediction experimenter possibility, treatment is had to reaction (" respondent ") or no reactionless to treating (" non-respondent ").According to this paper, use, the experimenter is accredited as for " respondent " for the treatment of or comprises described experimenter is accredited as respectively and may responds for described treatment for " the non-respondent " of described treatment, or may be reactionless for described treatment, and without the clearly prediction of the reaction of determining described experimenter.Available from one or more vesicas of experimenter or vesica colony for by assessment biomarker disclosed herein (as, the biomarker of listing in table 7), determining whether the experimenter is non-respondent or respondent for particular treatment.Select candidate therapeutic to the detection of the high or low expression level of biomarker or to the experimenter that the detection of the sudden change of biomarker can be used for for suffering from situation, such as drug intervention.Table 7 comprises the illustrative situation and to the drug intervention of these situations.This tabular has gone out the biomarker that affects described intervention effect.Can use method of the present invention to assess described biomarker, as, as circulating biological mark or the biomarker relevant to vesica.

Table 7: for the biomarker of situation and the example of drug intervention

Cancer

The biological marking of vesica can be used in the treatment diagnosis of cancer, such as identifying that suffering from cancered experimenter is respondent or non-respondent for specific cancer therapy possibility.Present method can be used for treating diagnosing cancer comprise those listed cancers of this paper (as, in " phenotype " part above).These cancers include but not limited to lung cancer, nonsmall-cell lung cancer, small cell lung cancer (comprises small cell carcinoma (oat-cell carcinoma), minicell/large cell carcinoma and Combination small cell carcinoma), colorectal carcinoma, mammary cancer, prostate cancer, liver cancer, carcinoma of the pancreas, the cancer of the brain, kidney, ovarian cancer, cancer of the stomach, melanoma, osteocarcinoma, cancer of the stomach, mammary cancer, glioma, glioblastoma multiforme, hepatocellular carcinoma, the papillary carcinoma kidney, squamous cell carcinoma of the head and neck, leukemia, lymphoma, myelomatosis or other noumenal tumour.

cancer: the biological marking

Can determine that the biological marking thinks that the experimenter provides treatment diagnosis.The biological marking of vesica can comprise one or more biomarkers, such as, but not limited to, any one or multiple biomarker as herein described, such as but not limited to, the biomarker of listing in Fig. 1,3,6,7,9-12,14-22,25-33,50-51,53-54,59 and 60.

The invention provides the several different methods of the identification of organism marking with characterizing cancers.This paper further provides biomarker, and it is assessed to identify the described biological marking.In one embodiment, the biological marking for prostate cancer comprises one or more following biomarker: EpCam, CD9, PCSA, CD63, CD81, PSMA, B7H3, PSCA, ICAM, STEAP and EGFR.In another embodiment, comprise EpCam+, CK+, CD45-vesica for the biological marking that prostate cancer is categorized as to castration resistance type (castration-resistant).In another embodiment, be used for the treatment of the biological marking of the vesica of diagnosing small cell lung cancer and comprise miR-451, miR-92a-2 ^*, miR-147 and/or miR-574-5p.In another embodiment again, for the biological marking of colorectal carcinoma treatment diagnosis, comprise one or more miR that are selected from miR-548c-5p, miR-362-3p, miR-422a, miR-597, miR-429, miR-200a and miR-200b.The biological marking that is used for the treatment of diagnosing cancer can comprise one or more in CA IX, CMET, VEGFR2, VEGF, vimentin, CD44v6, Ckit, Axl, RET (ret proto-oncogene), E cadherin and V cadherin.

cancer: nursing standard

The biological marking from the circulating biological mark in the sample of suffering from cancered experimenter (comprising the mark relevant to vesica) can be used for selecting candidate therapeutic for described experimenter.The method of the present invention that can provide according to this paper is determined the described biological marking.In some embodiments, candidate therapeutic comprises the nursing standard to described cancer.The described biological marking can be used for determining that the experimenter is non-reactor or reactor for particular treatment or nursing standard.Described treatment can be the treatment of cancer, such as radiotherapy, operative treatment, chemotherapy or its combination.Described cancer therapy can be that therapeutical agent is such as carcinostatic agent or chemotherapy regimen.Cancer therapy for method of the present invention includes but not limited to the treatment that table 8 is listed:

Table 8: cancer therapy

According to shown in table 8, cancer therapy comprises various operations and pharmacological agent.Carcinostatic agent comprises medicine, such as small molecules and biotechnological formulation.Method of the present invention can be used for identifying the biological marking comprise the circulating biological mark, and it can be used for the treatment of diagnostic purpose subsequently, such as monitor therapy effect, the experimenter is categorized as for reactor or the non-reactor for the treatment of or selects candidate therapeutic agent.The present invention can be used for for any cancer therapy provides the treatment diagnosis, and it includes but not limited to relate to the treatment diagnosis of the cancer therapy in table 8-10.But the method according to this invention is accredited as the cancer therapy of candidate therapeutic and includes but not limited to chemotherapeutics and the suitable combination arbitrarily thereof showing to list in 8-10.In one embodiment, described treatment is specific for the cancer of particular type, such as the listed treatment for prostate cancer, colorectal carcinoma, mammary cancer and lung cancer in table 8.In other embodiment, described treatment is specific for the tumour that shows the specific biological marking, and no matter it originates from, and shows the biological marking of the listed medicine Research of predicting markers of the listed mark of 9-10 or table 11 as comprised.

The invention provides the method for monitoring cancer therapy, it comprises with time-histories (such as before treatment and after treatment) or along with the time after treatment is identified a series of biological marking in the experimenter.The described biological marking and reference compare to determine the effect of described treatment.In one embodiment, described treatment is selected from table 8-10, such as radiation, operation, chemotherapy, biotherapy, neoadjuvant, adjuvant therapy or observation, waits for.Described reference can be from another individuality or group of individuals or from same subject.For example, have and indicate the experimenter of the front biological marking of cancer therapy can there is the biological marking of indicating state of health after successful treatment.On the contrary, the experimenter can have the biological marking of indication cancer after unsuccessful treatment.The described biological marking can compare to determine whether described experimenter's the biological marking indicates improvement, the deterioration or unchanged of situation in time.If described cancer worsens in time or be unchanged, may need other treatment.For example, can outside operation or radiotherapy, use hormonotherapy to have more invasive prostate cancer with treatment.Below one or more miR can be used for monitoring in the biological marking of prostate cancer therapy effect: hsa-miR-1974, hsa-miR-27b, hsa-miR-103, hsa-miR-146a, hsa-miR-22, hsa-miR-382, hsa-miR-23a, hsa-miR-376c, hsa-miR-335, hsa-miR-142-5p, hsa-miR-221, hsa-miR-142-3p, hsa-miR-151-3p, hsa-miR-21, hsa-miR-16.In following publication, one or more listed miR can be used for monitoring in the biological marking for the treatment of of GI road cancer: Albulescu etc., Tissular and soluble miRNAs for diagnostic and therapy improvement in digestive tract cancers, Exp Rev Mol Diag, 11:1,101-120.

In some embodiments, the invention provides and identify from the biological marking in experimenter's sample to select the method for candidate therapeutic agent.For example, the described biological marking can show that the relevant target of medicine undergos mutation or differential expression, shows therefrom that described experimenter may respond for particular treatment or reactionless.Candidate therapeutic can be selected from definite carcinostatic agent or therapeutical agent classification in table 8-10.In some embodiments, according to present method, definite candidate therapeutic is selected from the group that at least following treatment forms: 5 FU 5 fluorouracil, abarelix, alemtuzumab, aminoglutethimide, Anastrozole, asparaginase, acetylsalicylic acid, ATRA, azacitidine, rhuMAb-VEGF, Bexarotene, bicalutamide, calcitriol, capecitabine, carboplatin, celecoxib, Cetuximab, chemotherapy, cholecalciferol, cis-platinum, cytosine arabinoside, Dasatinib, daunorubicin, Decitabine, Dx, epirubicin, Tarceva, Etoposide, Exemestane, flutamide, fulvestrant, Gefitinib, gemcitabine, gonadorelin, goserelin, hydroxyurea, imatinib, irinotecan, lapatinibditosylate, letrozole, bright dried meat Li Te, the liposome Dx, medroxyprogesterone, megestrol, Magace, methotrexate, mitomycin, Abraxane, Sostatin, oxaliplatin, taxol, Victibix, pegaspargase, pemetrexed, pentostatin, Xarelto, Sutent, tamoxifen, taxanes, Temozolomide, toremifene, Herceptin, VBMCP and vincristine(VCR).

With selecting, candidate therapeutic is similar, and the present invention also provides the method for whether cancer being treated on earth of determining.For example, prostate cancer can be noninvasive disease, and it may not injure the experimenter in fact.The radiotherapy of following male sex hormone to remove (hormone reduction) is the standard method of the local advanced prostate cancer for the treatment of.The symptom of hormonotherapy comprises sexual dysfunction, hectic fever and hyposexuality.In addition, such as prostatectomy, such treatment can have such as sexual dysfunction or the such symptom of incontinence.Therefore, the invention provides the indication aggressive of cancer or progress (as by stages or grade) the biological marking.Non-Invasive cancer or localization cancer may be without treatments immediately but can be observed, as, to " observe wait for " of prostate cancer.And aggressive or late period focus need side by side carry out more positive treatment plan.

The example of detectable biomarker and can select or the example of the therapeutical agent that may be avoided is listed in table 9.For example,, for suffering from experimenter's identification of organism marking of prostate cancer, the level that the wherein said biological marking comprises androgen receptor (AR).Cross expressing of AR or determining that excessive generation (such as the high level of mRNA level or protein level in vesica) is candidate therapeutic that described experimenter is provided.These treatments comprise the medicine that is used for the treatment of described experimenter, such as bicalutamide, flutamide, bright dried meat Li Te or goserelin.Therefore described experimenter is confirmed as the reactor for bicalutamide, flutamide, bright dried meat Li Te or goserelin.In another illustrative example, detect high-caliber BCRP mRNA, protein or both in the vesica of the experimenter from suffering from NSCLC.Described experimenter thereby can be confirmed as the non-reactor of medicine cis-platinum and carboplatin, or described medicine is considered in described experimenter for treatment NSCLC effect lower than other medicines and is not selected for the described experimenter for the treatment of.Can in the vesica available from the experimenter, assess following any biomarker, and described biomarker can be for including but not limited to the form of one or more nucleic acid, polypeptide, peptide or plan peptide material.In another illustrative example again, the sudden change of one or more in KRAS, BRAF, PIK3CA and/or c-kit can be used for selecting candidate therapeutic.For example, in the patient sudden change of KRAS or BRAF can show Cetuximab and/or Victibix may be in the described patient for the treatment of poor efficiency comparatively.

Table 9: the example of biomarker, pedigree and medicine

Detectable biomarker and can being selected or the example of other therapeutical agent that may be avoided is listed in table 10 according to the described biological marking.For example, for suffering from cancered experimenter, crossing of ADA detected and express for described experimenter is categorized as to the respondent for pentostatin in the vesica from the experimenter, or for pentostatin being accredited as to the medicine that is used for the treatment of described experimenter.In another embodiment, for suffering from cancered experimenter, crossing of BCRP detected and express for described experimenter being categorized as to the non-respondent for cis-platinum, carboplatin, irinotecan and Hycamtin in the vesica from described experimenter, this means that cis-platinum, carboplatin, irinotecan and Hycamtin are accredited as the medicine for the non-the best of the described experimenter for the treatment of.

Table 10: the example of biomarker, medicine and resistance

The further medicine used in embodiment of the present invention is associated is found in rule the U.S. Patent application 12/658,770 of submitting on February 12nd, 2010; International PCT patent application PCT/US2010/000407 that on February 11st, 2010 submits to; International PCT patent application PCT/US2010/54366 that on October 27th, 2010 submits to; And the U.S. Provisional Patent Application 61/427,788 of submission on December 28th, 2010; All application is incorporated to this paper in full by reference.For example,, referring to " Table 4:Rules Summary for Treatment Selection " in PCT/US2010/54366.

The associated target of medicine can be the part of the biological marking for the treatment diagnosis is provided arbitrarily.Comprise and can use the target that therapeutical agent (such as small molecules and biological products) regulated " but medication target (druggable target) " to be included in the candidate in the biological marking of the present invention.The associated target of medicine also can comprise the biomarker that can cause the resistance for the treatment of, shown in table 9 and 10.The described biological marking can based on gene (as, DNA sequence dna) and/or gene product (as mRNA or protein) or as described in the associated target of medicine.Whether these nucleic acid and/or polypeptide can exist with regard to it, but the applicable feature in level or amount, activity, sudden change, sequence, haplotype, rearrangement, copy number or other measurement features is carried out somatotype.Described gene or gene product can join with the vesica cluster correlation, for example, and as vesica surface marker or vesica useful load.In one embodiment, the invention provides the method for the treatment of diagnosing cancer, it comprises the identification of organism marking (its comprise the associated targets of one or more medicines have situation or a level) and selects candidate therapeutic agent according to the described biological marking.The associated target of described medicine can be circulating biological mark, vesica or vesica associated biomolecule mark.Because the associated target of medicine may be irrelevant with tissue or cell source, therefore the biological marking that comprises the associated target of medicine can be used for providing the treatment diagnosis for any proliferative disease, such as the cancer from various different anatomic origins, comprise the cancer of unknown source, such as CUPS.

Use the associated target of medicine of method assessment of the present invention to include but not limited to: ABCC1, ABCG2, ACE2, ADA, ADH1C, ADH4, AGT, AR, AREG, ASNS, BCL2, BCRP, BDCA1, β III tubulin, BIRC5, B-RAF, BRCA1, BRCA2, CA2, caveolin, CD20, CD25, CD33, CD52, CDA, CDKN2A, CDKN1A, CDKN1B, CDK2, CDW52, CES2, CK 14, CK 17, CK 5/6, c-KIT, c-Met, c-Myc, COX-2, cyclin D1, DCK, DHFR, DNMT1, DNMT3A, DNMT3B, CAM 120/80, ECGF1, EGFR, the EML4-ALK syzygy, EPHA2, epiregulin, ER, ERBR2, ERCC1, ERCC3, EREG, ESR1, FLT1, folacin receptor, FOLR1, FOLR2, FSHB, FSHPRH1, FSHR, FYN, GART, GNRH1, GNRHR1, GSTP1, HCK, HDAC1, hENT-1, Her2/Neu, HGF, HIF1A, HIG1, HSP90, HSP90AA1, HSPCA, IGF-1R, IGFRBP, IGFRBP3, IGFRBP4, IGFRBP5, IL13RA1, IL2RA, KDR, Ki67, KIT, K-RAS, LCK, LTB, lymphotoxin-beta-receptor, LYN, MET, MGMT, MLH1, MMR, MRP1, MS4A1, MSH2, MSH5, Myc, NFKB1, NFKB2, NFKBIA, ODC1, OGFR, p16, p21, p27, p53, p95, PARP-1, PDGFC, PDGFR, PDGFRA, PDGFRB, PGP, PGR, PI3K, POLA, POLA1, PPARG, PPARGC1, PR, PTEN, PTGS2, RAF1, RARA, RRM1, RRM2, RRM2B, RXRB, RXRG, SPARC, SRC, SSTR1, SSTR2, SSTR3, SSTR4, SSTR5, Survivin, TK1, TLE3, TNF, TOP1, TOP2A, TOP2B, TS, TXN, TXNRD1, TYMS, VDR, VEGF, VEGFA, VEGFC, VHL, YES1 and ZAP70, and their arbitrary combination.Comprise that a kind of or biological marking combination in these marks can be used for characterizing according to the present invention phenotype, such as the treatment diagnosis is provided.Known these marks play a role in the effect of multiple chemotherapeutics antagonism proliferative disease.Therefore, can be assessed to be independent of cancer source or type selecting candidate therapeutic for described cancer to described mark.In one embodiment, the invention provides the method for selecting candidate therapeutic agent for cancer, it comprises identifies the level comprise the associated targets of one or more medicines or the biological marking of existence, and selects candidate therapeutic agent according to its effect of expection for the patient with described biological marking.The associated targets of described one or more medicines can be one of targets shown in listed or table 9-11 above.In some embodiments, assess at least 2,3,4,5,6,7,8,9,10,12,15,20,25,30,35,40,45 in the associated targets of described one or more medicines or at least 50 kinds.The associated targets of described one or more medicines can nucleic acid (as DNA, mRNA) or the form of protein relevant to vesica, for example, as vesica surface marker or vesica useful load.In some embodiments, existence or the level of the interactional microRNA of the associated target of known and described one or more medicines have been assessed, wherein the high-level microRNA of the associated targets of described one or more medicines of known inhibition can mean low expression of the associated targets of described one or more medicines, and pointed out thus may be lower to the reaction of the treatment for the associated target of described medicine.The associated target of described one or more medicines can be the circulating biological mark.The associated target of described one or more medicines can be assessed in tissue sample.Can compare with reference point and determine described expection effect by the existence by the associated target of described one or more medicines and level, wherein higher than the level of described reference, pointing out described experimenter may be the respondent.Can use sorting algorithm to determine described expection effect, wherein by by known for described candidate therapeutic, be that the biological marking of the associated targets of one or more medicines in respondent or non-respondent's experimenter is compared and trains described sorter.The associated targets of described one or more medicines are associated with the molecule of suitable candidate's target to be shown in the table 9-10 of this paper and the U.S. Patent application 12/658,770 of submission on February 12nd, 2010; International PCT patent application PCT/US2010/000407 that on February 11st, 2010 submits to; International PCT patent application PCT/US2010/54366 that on October 27th, 2010 submits to; And among the U.S. Provisional Patent Application 61/427,788 of submission on December 28th, 2010; All application is incorporated to this paper in full by reference.

Table 11 provides many treatment diagnosis target target genes that the method according to this invention analyzed and corresponding protein code name and the list of title.According to those skilled in the art, understand, gene and protein develop out a large amount of substituting titles in scientific and technical literature.Therefore, enumerating in table 11 comprised illustrative but the gathering of non-limit.Gene another name and the further list of describing can be used multiple online database to obtain, and it comprises GeneCards

(www.genecards.org), HUGO Gene Nomenclature (www.genenames.org), Entrez Gene (www.ncbi.nlm.nih.gov/entrez/query.fcgi? db=gene), UniProtKB/Swiss-Prot (www.uniprot.org), UniProtKB/TrEMBL (www.uniprot.org), OMIM (www.ncbi.nlm.nih.gov/entrez/query.fcgi? db=OMIM), GeneLoc (genecards.weizmann.ac.il/geneloc/) and Ensembl (www.ensembl.org).Usually, code name and title that gene code name hereinafter and title are checked and approved corresponding to HUGU usually, and the protein title is the title by the UniProtKB/Swiss-Prot suggestion.Designate commonly used also is provided.In the situation that the protein title means precursor, also comprised ripe protein.In the application's full text, gene and protein code name are used interchangeably and described implication can be known by inference from context where necessary.

Table 11: for gene and the related protein of cancer therapy diagnosis

Known gene and the gene product that works in cancer and can be contained in the biological marking of the present invention includes but not limited to 2AR, disintegrin, Tiroidina and retinoid receptor activator (ACTR), ADAM 11, steatogenesis supressor (ADIF), α 6 integrin subunits, α V integrin subunit, α-catenin, mammary cancer amplified material 1 (AIB1), mammary cancer amplified material 3 (AIB3), mammary cancer amplified material 4 (AIB4), amyloid precursor protein secretase (APPS), AP-2 γ, APPS, ATP linking frame transporter (ABCT), placenta specific (ABCP), ATP linking frame subfamily C member (ABCC1), BAG-1, BASIGIN (BSG), BCEI, B cell differential factor (BCDF), B cell leukemia 2 (BCL-2), B-cell stimulating factor-2 (BSF-2), BCL-1, the BCL-2 X protein (BAX) of being correlated with, BCRP, β 1 integrin subunit, β 3 integrin subunits, β 5 integrin subunits, β-2 Interferon, rabbit, beta-catenin, beta-catenin, bone sialoprotein (BSP), mammary cancer estrogen-induced type sequence (BCEI), mammary cancer resistance protein (BCRP), mammary cancer 1 type (BRCA1), mammary cancer 2 types (BRCA2), mammary cancer extension increasing sequence 2 (BCAS2), cadherin, E-cadherin-11, the cadherin associated protein, Calcitonin Receptor (CTR), calcium placental protein (CAPL), calcyclin (CALCYCLIN), CALLA, CAM5, CAPL, carcinomebryonic antigen (CEA), catenin, α 1, cathepsin B, cathepsin D, cathepsin K, proteinase cathepsin L2, kethepsin O, kethepsin O1, kethepsin V, CD10, CD146, CD147, CD24, CD29, CD44, CD51, CD54, CD61, CD66e, CD82, CD87, CD9, CEA, cellular retinol binding protein 1 (CRBP1), C-ERBB-2, CK7, CK8, CK18, CK19, CK20, tight junction protein-7, c-Met, collagenase, inoblast, collagenase, interstitial, collagenase, CALLA (CALLA), connexin 26 (Cx26), connect protein 43 (Cx43), the cortex Actin muscle, COX-2, CTLA-8, CTR, CTSD, cyclin D1, COX-2, CK18, Cyfra21-1, CK8, the cytotoxic T lymphocyte serine easterase 8 (CTLA-8) of being correlated with, differentiation suppresses active (DIA), DNA cloning thing 1 (DAM1) in mammary cancer, DNA topoisomerase II α, DR-NM23, CAM 120/80, the emmprin factor (EMMPRIN), EMS1, vascular endothelial growth factor (ECGR), thrombocyte source property (PD-ECGF), enkephalin, EGF-R ELISA (EGFR), EPISIALIN, EMA (EMA), ER-α, ERBB2, ERBB4, ER-β, ERF-1, red is enhanced activity (EPA), ESR1, estrogen receptor alpha, estrogen receptor-beta, ETS-1, the emmprin factor (EMMPRIN), fibronectin receptor, beta polypeptides (FNRB), fibronectin receptor beta subunit (FNRB), FLK-1, GA15.3, GA733.2, Gal-3, γ-connection albumen, gap junction protein (26kDa), gap junction protein (43kDa), gap junction protein α-1 (GJA1), gap junction protein β-2 (GJB2), GCP1, gelatin enzyme A, Gelatinase B, gelatinase (72kDa), gelatinase (92kDa), gum inhibitor (GLIOSTATIN), glucocorticoid receptor interaction protein 1 (GRIP1), glutathione-S-transferase p, GM-CSF, granulocyte chemoattractant protein 1 (GCP1), granulocyte-macrophage colony stimutaing factor, growth factor receptors binding substances-7 (GRB-7), GSTp, HAP, heat shock protein 70 (HSC70), heat stable antigen, pHGF (HGF), hepatocyte growth factor receptor (HGFR), hepatocyte-stimulating factor III (HSF III), HER-2, HER2/neu, HERMES antigen, HET, HHM, body fluid malignant hypercalcemia (HHM), ICERE-1, INT-1, intercellular adhesion molecule-1 (ICAM-1), gamma-interferon inducible factor (IGIF), il-1 α (IL-1A), il-1 β (IL-1B), interleukin-11 (IL-11), interleukin-17 (IL-17), IL-18 (IL-18), interleukin-6 (IL-6), interleukin-8 (IL-8), estrogen receptor expression negative correlation 1 (ICERE-1), KAI1, KDR, CK8, Keratin 18, Keratin 19, KISS-1, leukaemia inhibitory factor (LIF), LIF, inflammatory breast cancer disappearance thing (LIBC), LOT (" transforming disappearance "), lymphocyte homing receptor, macrophage colony stimulating factor, MAGE-3, mammaglobin, MASPIN, MC56, M-CSF, MDC, MDNCF, MDR, melanoma cell adhesion molecule (MCAM), film Zinc metalloproteinase (MME), the film neutral endopeptidase (NEP) of being correlated with, be rich in cysteine protein (MDC), transfer protein (METASTASIN) (MTS-1), MLN64, MMP1, MMP2, MMP3, MMP7, MMP9, MMP11, MMP13, MMP14, MMP15, MMP16, MMP17, moesin, monocyte arginine-serpin, monocyte source neutrophilic chemotactic factor, monocyte source property Type 1 plasminogen activator inhibitor, MTS-1, MUC-1, MUC18, Saliva Orthana sample cancer associated antigen (MCA), MUCIN, MUC-1, Mdr-p 1 (MDR, MDR1), multidrug-associated protein 1 (MRP, MRP-1), the N-cadherin, NEP, NEU, neutral endopeptidase, neutrophil activation peptide 1 (NAP1), NM23-H1, NM23-H2, NME1, NME2, nuclear receptor coactivator 1 (NCOA-1), nuclear receptor coactivator-2 (NCOA-2), nuclear receptor coactivator-3 (NCOA-3), nucleoside diphosphate kinase A (NDPKA), rmNDPK-B (NDPKB), oncostatinM (OSM), ornithine decarboxylase (ODC), osteoclast differentiation factor (ODF), osteoclast differentiation factor acceptor (ODFR), osteonectin (OSN, ON), osteopontin (OPN), ocytocin receptor (OXTR), p27/Kip1, p300/CBPCOINTEGRATOR associated protein (P/CIP), P53, p9Ka, PAI-1, PAI-2, parathyroid adenoma 1 (PRAD1), Rat parathyroid hormone 1-34 sample hormone (PTHLH), parathyroid hormone-related peptide (PTHrP), the P-cadherin, PD-ECGF, PDGF, the reactive uromucoid (PUM) of peanut, p-glycoprotein (P-GP), PGP-1, PHGS-2, PHS-2, PIP, plakoglobin, Type 1 plasminogen activator inhibitor (1 type), Type 1 plasminogen activator inhibitor (2 type), plasminogen activator (tissue-type), plasminogen activator (urokinase type), platelet glycoprotein IIIa (GP3A), PLAU, pleomorphic adenoma gene sample 1 (PLAGL1), multiform mucins (PEM), PRAD1, progesterone receptor (PGR), the progestogen resistance, prostaglandin endoperoxides synthetic enzyme-2, prostaglandin G/H sythase-2, prostaglandin H synthase-2, pS2, PS6K, psoriasin (PSORIASIN), PTHLH, PTHrP, RAD51, RAD52, RAD54, RAP46, the acceptor co-activation factor 3 (RAC3) of being correlated with, estrogen receptor activity inhibition (REA), S100A4, S100A6, S100A7, S6K, SART-1, skeleton attachment element B (SAF-B), scattering factor (SF), secretor type phosphoric acid albumen-1 (SPP-1), secretory protein, rich acidic is containing halfcystine (SPARC), STANNICALCIN, the steroid receptor co-activation factor 1 (SRC-1), the steroid receptor co-activation factor 2 (SRC-2), the steroid receptor co-activation factor 3 (SRC-3), steroid receptor RNA activator (SRA), stromelysin-1, Stromelysin-3, tenascin-C (TN-C), testis specific protein enzyme 50, thrombostondin I, thrombostondin II, thymidine phosphorylase (TP), Thyroid Hormone Receptors activated molecule 1 (TRAM-1), tight junction protein 1 (TJP1), TIMP1, TIMP2, TIMP3, TIMP4, tissue factor (TF), tissue-type plasminogen activator, TN-C, TP53, tPA, the factor 2 (TIF2) in the middle of transcribing, trefoil factor 1 (TFF1), TSG101, TSP-1, TSP1, TSP-2, TSP2, TSP50, tumour cell collagenase stimulating factor (TCSF), the Tumor-assaciated mucins, uPA, uPAR, urokinase, urokinase type plasminogen activator, urokinase type plasminogen activator acceptor (uPAR), uvomorulin (UVOMORULIN), vascular endothelial growth factor, VEGF R2 (VEGFR2), VEGF-A, vascular permeability factor, VEGFR2, utmost point T in late period cell antigen β (VLA-β), vimentin, Vitronectic receptor α polypeptide (VNRA), Vitronectic receptor, vWF ELISA, VPF, VWF, WNT-1, ZAC, ZO-1 and locking band-1.Described gene and/or gene product can be parts that is used for the treatment of the biological marking of diagnosing cancer.

As explanation, can select for the experimenter who suffers from nonsmall-cell lung cancer treatment.Can assess one or more biomarkers from the vesica from described experimenter, such as, but not limited to, cross complementary group 1 (ERCC1), p53, Ras, p27, III beta tubulin, mastocarcinoma gene 1 (BRCA1), mastocarcinoma gene 1 (BRCA2) and ribonucleoside reductase enzyme courier 1 (RRM1) are repaired in EGFR, excision.According to one or more features of described one or more biomarkers, described experimenter can be defined as to respondent or non-respondent for treatment (such as, but not limited to Tarceva, carboplatin, taxol, Gefitinib or its combination).

In another embodiment, can be the experimenter who suffers from colorectal carcinoma and select treatment, and can be from the assessment of the vesica from described experimenter biomarker, such as, but not limited to K-ras.According to one or more features of described one or more biomarkers, described experimenter can be defined as to respondent or non-respondent for treatment (such as, but not limited to Victibix, Cetuximab or its combination).

In another embodiment, can select for the experimenter who suffers from mammary cancer treatment.Can assess one or more biomarkers from the vesica from described experimenter, such as, but not limited to HER2, topoisomerase II α, estrogen receptor and progestin acceptor.According to one or more features of described one or more biomarkers, described experimenter can be defined as to respondent or non-respondent for treatment (such as, but not limited to Herceptin, anthracycline, Taxan, methotrexate, Fluracil or its combination).

As described, the biological marking that is used for the treatment of diagnosing cancer can comprise the analysis of one or more biomarkers (it can be protein or nucleic acid), comprises mRNA or microRNA.Described biomarker can be in body fluid, detected, and/or the biomarker relevant to vesica can be detected, as, as vesica antigen or vesica useful load.In an illustrative example, the described biological marking is for being accredited as respondent or the non-respondent for tyrosine kinase inhibitor by the patient.Described biomarker can be the WO/2010/121238 that is entitled as " METHODS AND KITS TO PREDICT THERAPEUTIC OUTCOME OF TYROSINE KINASE INHIBITORS " submitted on April 19th, 2010; Or one or more biomarkers of describing in the WO/2009/105223 that is entitled as " SYSTEMS AND METHODS OF CANCER STAGING AND TREATMENT " of submission on February 19th, 2009, two applications all are incorporated to this paper in full by reference.

In one aspect, the invention provides the mensuration experimenter responds or unresponsive method for the tyrosine kinase inhibitor possibility, described method is included in from identifying in the vesica colony of described experimenter's sample and wherein with reference to comparing the differential expression of described one or more biomarkers in described sample shows that described experimenter is respondent or non-respondent for described tyrosine kinase inhibitor by one or more biomarkers.In one embodiment, described one or more biomarkers comprise miR-497, and it is respondent's (that is, for described tyrosine kinase inhibitor sensitivity) that described experimenter is indicated in the reduction that wherein miR-497 expresses.In another embodiment, described one or more biomarkers comprise one or more in miR-21, miR-23a, miR-23b and miR-29b, it may be non-respondent (that is, described tyrosine kinase inhibitor being had to resistance) that described experimenter is indicated in the rise of wherein said microRNA.In some embodiments, described one or more biomarkers comprise one or more in hsa-miR-029a, hsa-let-7d, hsa-miR-100, hsa-miR-1260, hsa-miR-025, hsa-let-7i, hsa-miR-146a, hsa-miR-594-Pre, hsa-miR-024, FGFR1, MET, RAB25, EGFR, KIT and VEGFR2.In another embodiment, described one or more biomarkers comprise FGF1, HOXC10 or LHFP, wherein said biomarker to indicate described experimenter than high expression level be non-respondent (that is, described tyrosine kinase inhibitor being had to resistance).Described method can be used for measuring the susceptibility of cancer for described tyrosine kinase inhibitor, for example, and non-small cell lung cancer cell, kidney or GIST.Described tyrosine kinase inhibitor can be Tarceva, ZD6474, Sutent and/or Xarelto, or by other inhibitor of similar effect mechanism works.Tyrosine kinase inhibitor comprises any medicine that suppresses the effect of one or more Tyrosylprotein kinases with specificity or non-specific mode.Tyrosine kinase inhibitor comprises small molecules, antibody, peptide or suitable entity arbitrarily, its directly, indirectly, allosteric ground or suppress the phosphorylation of tyrosine residues with any alternate manner.The particular instance of tyrosine kinase inhibitor comprises N-(trifluoromethyl)-5-methyl-isoxazole-4-methane amide, 3-[(2,4-dimethyl pyrrole-5-yl) methylene radical) Indolin-2-one, 17-(allylamino)-17-AAG, 4-(the chloro-4-fluorophenyl of 3-amino)-7-methoxyl group-6-[3-(4-morpholinyl) propoxy-] q-quinazoline, N-(3-ethynyl phenyl)-6,7-bis-(2-methoxy ethoxy)-4-quinazoline amine, BIBX1382,2,3,9,10,11,12-six hydrogen-10-(methylol)-10-hydroxyl-9-methyl-9,12-epoxy-y-1H- [1,2,3-fg:3 ', 2 ', 1 '-kl] pyrrolo-[3,4-i] [1,6] benzo diamino Fang Xin-1-ketone, SH268, genistein, STI571, CEP2563, 4-(3-chloro-phenyl-amino)-5, and 6-dimethyl-7H-pyrrolo-[2,3-d) the pyrimidine methane sulfonate, 4-(the bromo-4-hydroxy phenyl of 3-) amido-6,7-dimethoxy quinazoline, 4-(4 '-hydroxy phenyl) amido-6,7-dimethoxy quinazoline, SU6668, STI571A, N-4-chloro-phenyl--4-(4-pyridylmethyl)-1-phthalazines amine (phthalazinamine), N-[2-(diethylin) ethyl]-5-[(z)-(5-fluoro-1,2-dihydro-2-oxo--3H-indoles-3-subunit) methyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide (being commonly referred to Sutent), the chloro-3-of A-[-A-[[4-(trifluoromethyl) phenyl]-carbamyl amino]-phenoxy group }-N-methyl-pyridine-2-carboxamide (being commonly referred to Xarelto), EMD121974 and N-(3-ethynyl phenyl)-6, two (2-methoxy ethoxy) quinazolines of 7--4-amine (usually claiming Tarceva).In some embodiments, described tyrosine kinase inhibitor has and suppresses active for EGF-R ELISA (EGFR), VEGFR, PDGFR β and/or FLT3.

Therefore, can be imprinted as and suffer from cancered experimenter and select treatment according to the biology of identifying by method of the present invention.Therefore, the described biological marking can comprise circulating biological mark existence or the level of (comprising microRNA, vesica or any available vesica associated biomolecule mark).

Cardiovascular

The assessment vesica can be used for the treatment diagnosis of cardiovascular status, imbalance or disease.Cardiovascular status includes but not limited to, chronic rheumatic heart disease, essential hypertension, ischemic heart disease, disease of pulmonary circulation, heart trouble, cerebrovascular disease, artery, arteriole and disease of capillaries and vein and lymphsystem disease.Chronic rheumatic heart disease includes but not limited to, mitral valve disease, aortic valve disease, mitral valve and aortic valve disease and other endocardium structure disease.High blood pressure disease includes but not limited to, essential hypertension, malignant hypertension, benign hypertension, not clear hypertension, hypertensive heart disease, hypertensive renal disease, the not clear concurrent renal failure of hypertensive renal disease, hypertensive cerebral heart and kidney disease, pernicious renovascular hypertension and optimum renovascular hypertension.Ischemic heart disease includes but not limited to Acute Myocardial Infarction, acute front lateral myocardium infraction, acute anterior myocardial infarction, acute lower outside myocardial infarction, acute myocardial infarction of inferoposterior wall, acute myocardial infarction of inferior wall, acute other inferior myocardial infarction, acute other lateral myocardial infarction, acute strictly posterior myocardial infarction, acute subendocardiac muscle myocardial infarction, acute spec myocardial infarction, acute not clear myocardial infarction, postmyocardial infarction syndrome, middle coronary syndrome, remote myocardial infarction, stenocardia, angina pectoris decubitus, variant angina pectoris, coronary atherosclerosis, arterio-cardiac aneurysm and interlayer, the heart wall knurl, coronary aneurysm, coronary artery dissection and not clear chronic ischemic heart disease.

Disease of pulmonary circulation includes but not limited to, the disease of pulmonary circulation, acute pulmonary heart disease, non-iatrogenic lung embolism, chronic cardiopulmonary disease and not clear chronic cardiopulmonary disease.Heart trouble includes but not limited to acute pericarditis, other and not clear acute pericarditis, acute nonspecific pericarditis, acute and subacute endocarditis, the acute bacterial endocarditis acute myocarditis, other and non-specific acute myocarditis, congenital myocarditis, other pericardial disease, other endocardial disease, the mitral valve obstacle, aortic valve valve obstacle, the tricuspid valve obstacle, pulmonary valve valve obstacle, myocardosis, hypertrophic obstructive cardiomyopathy, conductive impairment, third degree A-V block, first degree A-V block, Mohs II atrioventricular block, Wen's atrioventricular block, left bundle branch block, right bundle branch block, hole heart block, abnormal Atrioventricular Conduction excites, preexcitation syndrome, heart arrhythmias, paroxysmal supraventricular tachycardia, atrial fibrillation and fluttering, atrial fibrillation, auricular flutter, ventricular fibrillation and fluttering, ventricular fibrillation, asystole, premature beat, other specific arrhythmia, sick sinus syndrome, sinus bradycardia, not clear heart disorder, gallop rhythm, in heart failure, congestive heart failure, acute lung edema, systole is not clear in heart failure, acute systolic heart failure, chronic systolic heart failure, diastole is not clear in heart failure, the diastole chronic heart failure, merge not clear heart failure and cardiac hypertrophy.

Cerebrovascular disease includes but not limited to, subarachnoid hemorrhage, intracerebral hemorrhage, other and not clear intracranialing hemorrhage, intracranial hemorrhage, occlusion of precerebral artery and narrow, basilar artery occlusion and narrow, carotid obturation and narrow, vertebrarterial obturation and narrow, cerebral artery occlusion, cerebral thrombosis, without the cerebral infarction cerebral thrombosis, cerebral infarction cerebral thrombosis shape, cerebral embolism, without the cerebral infarction cerebral embolism, the cerebral infarction cerebral embolism, transient ischemic attack, arteria basilaris syndrome, vertebral artery syndrome, subclavian steal syndrome, the vertebra arteria basilaris syndrome, transient ischemic attack, acute indefinite cerebrovascular disease, indefinite cerebrovascular disease, cerebral atherosclerosis, other broad sense ischemic cerebrovascular, hypertensive encephalopathy, uncracked cerebral aneurysm, cerebral arteritis, moyamoya, the non-gathering thrombosis of dlural sinus, transient global amnesia, the anaphase effect of cerebrovascular disease, cognitive defect, speech and language defect, not clear sound language and language defect, aphasia, dysphagia, other speech and language defect, hemiplegia/hemiparesis, not clear lateral deviation paralysis, habitual lateral deviation paralysis, non-habitual lateral deviation paralysis, upper limbs monoplegia, lower limb monoplegia, other paralytic syndrome, other later stage impact of cerebrovascular disease, the cerebrovascular disease apraxia, the cerebrovascular disease dysphagia, facial paralysis, ataxia and dizzy.

Artery, arteriole and disease of capillaries include but not limited to atherosclerosis, atherosclerosis of renal artery, autologous artery of extremity atherosclerosis, intermittent claudication, atherosis without the ulcer artery of extremity, non-heart and brain atherosclerosis, aortic aneurysm, dissection of aorta, Ruptured Abdominal Aortic Aneurysm, the crack-free abdominal aortic aneurysm, not clear aortic aneurysm, other aneurysma, other peripheral vascular disease, Raynaud's syndrome (raynaud ' s syndrome), thromboangiitis obliterans, other artery dissection, the internal carotid artery interlayer, the iliac artery interlayer, Renal artery interlayer, the vertebral artery interlayer, other artery dissection, erythromelalgia, not clear peripheral vascular disease, arterial thrombosis and thrombosis, polyarteritis nodosa and conditions associated, polyarteritis nodosa, mucocutaneous lymphnode syndrome/acute febrile mucocutaneous lymph-node syndrome, allergic angiitis, Goodpastures syndrome, lethal midline granuloma, the Wei Genashi granuloma, giant cell arteritis, thrombotic microangiopathy, the Takayasu disease, other artery and arteriole disease, acquired arterio venous fistula, not clear arteritis, vasculitis, the Non-cancerous naevus vascularis.

Vein and lymphsystem disease include but not limited to, phlebitis and thrombophlebitis, the deep femoral vein thrombosis, the venous thrombosis of other shank vein, the phlebitis at other position of superficial veins of upper limb, not clear thrombophlebitis, pylethrombosis, other venous thrombosis and thrombosis, not clear venous thrombosis, the near-end venous thrombosis, the far-end venous thrombosis, not clear venous thrombosis, Varicose veins of lower extremity, without ulcer varix, NIP varix, without ulcer NIP varix, asymptomatic varix, hemorrhoid, Internal hemorrhoids without complication, external hemorrhoids without complication, thrombosed external hemorrhoids, other position varix hemorrhoid, esophageal varicosis is without hemorrhage, esophageal varicosis is without hemorrhage, varicocele, the non-infectious obstacle of lymphatic vessel, postmastectomy lymphedema syndrome, ypotension, postural hypotension, iatrogenic ypotension, other recycle system obstacle, other particular cycle system disorders and not clear venous insufficiency.

Other example of heart includes but not limited to, coronary occlusion (for example, because blood fat/cholesterol deposits, scavenger cell/inflammatory cell are raised, plaque rupture, thrombosis, platelet deposition or vascellum endometrial hyperplasia cause or relative); Ischemic syndrome (that for example, cause due to myocardial infarction, stable angina pectoris, unstable angina pectoris, coronary restenosis or reperfusion injury or relative); Myocardosis (that for example, cause due to ischemic syndrome, cardiotoxin, infection, hypertension, metabolic disease (as uremia, vitamin B1 deficiency or glycogenosis), radiation, neuromuscular disease, wetting property disease (as sarcoidosis, hemochromatosis, amyloidosis, FabryShi disease or Hurler Cotard), wound or congenital reason or relative); Heart disorder or arrhythmia (that for example, due to ischemia syndrome, cardiotoxin, Zorubicin, infection, hypertension, metabolic disease, radiation, neuromuscular disease, wetting property disease, wound or congenital reason, cause or relative); Infect (for example, by pathogenic agent, being caused, as bacterium, virus, fungi or parasite); Inflammatory conditions (for example, to myocarditis, pericarditis, endocarditis, immunity heart, repelling inflammatory situation relevant or that caused by one of congenital, autoimmunity or connective tissue disease).

cardiovascular: the biological marking

Can assess the biological marking of vesica and diagnose for the experimenter provides treatment.The biological marking of described vesica can comprise one or more biomarkers, such as, but not limited to, any one or more biomarker as herein described, as but be not limited to, the biomarker of listing in Figure 24, miR-21, miR-129, miR-212, miR-214, miR-134 and other, such as the biomarker be described in US publication No.2010/0010073.

cardiovascular: nursing standard

The biological marking of the vesica of the sample of the experimenter from suffering from heart, imbalance or disease, vesica amount or both are measured and can be used for selecting nursing standard for described experimenter.Described nursing standard can comprise therapeutical agent or process (as, angioplasty).The example of therapeutical agent includes but not limited to, vascularization promotor (as, vascular endothelial growth factor, nitrogen protoxide releasing agent or generation agent, fibroblast growth factor, platelet derived growth factor, interleukin 6, MCP 1, rHuGM-CSF, transforming growth factor-beta), antithrombotic agent (as, acetylsalicylic acid, heparin, PPACK, enoxaparin, r-hirudin), anti-coagulant, microbiotic, anti-platelet agents, thrombolytics (as, tissue-type plasminogen activator), antiproliferative, anti-inflammatory agent, the medicine that suppresses hyperplasia, the medicine that suppresses restenosis, the smooth muscle cell inhibitor, somatomedin, growth factor receptor inhibitors, cell adhension inhibitors, chemotherapeutics and their combination.

For example, detection to one or more microRNA biomarkers (such as miR-21, miR-129, miR-212, miR-214, miR-134 or their combination) can be used for characterizing myocardial hypertrophy and/or heart failure, and it provides the treatment diagnosis for described myocardial hypertrophy.Described treatment diagnosis can comprise the selection such as using the therapy that vascularization promotor is such.Other example for the treatment of comprises and is used for the treatment of cholesterol and the abnormal treatment of triglyceride level blood level, such as listed in table 12.

Table 12: the example of the drug categories for the treatment of for cardiovascular status

In one embodiment, can select for the experimenter who suffers from peripheral arterial disease treatment.Can assess one or more biomarkers by the vesica from described experimenter, such as, but not limited to, c reactive protein (CRP), serum amyloid A protein (SAA), interleukin 6, intracellular adhesion molecule (ICAM), blood vessel adhesion molecule (VCAM), CD40L, Fibrinogen, scleroproein DDi, fibrinopeptide A, the von Willibrand factor, tissue-type plasminogen activator's antigen (t-PA), factor VII, Prothrombin fragment 1, Ox LDL (oxLDL) and lipoprotein A.According to one or more features of described one or more biomarkers, described experimenter can be defined as to respondent or non-respondent for treatment (such as, but not limited to Zarator, Simvastatin, superstatin, Pravastatin, fluvastatin, lovastatin or its combination).

In another embodiment, can select for the experimenter who suffers from arrhythmia treatment.Can assess one or more biomarkers by the vesica from described experimenter, such as, but not limited to SERCA, AAP, connection albumen 40, connection protein 43, ATP responsive type potassium channel, Kv1.5 passage and Activated By Acetylcholine potassium channel.One or more features according to described one or more biomarkers, described experimenter can be defined as to respondent or non-respondent for treatment, described treatment such as, but be not limited to, disopyramide, flecainide, lignocaine, mexiletine, Moracizine, procainamide, propafenone hydrochloride, quinine, tocainide, acebutolol, betaxolol, bisoprolol, carvedilol, esmolol, metoprolol, nadolol, Proprasylyte, sotalol, timolol, amiodarone, Azimilide, Bepridil, P162a, ibutilide, tedisamil, sulphur nitrogen

ketone, verapamil, Azimilide, Dronedarone, amiodarone, PM101, ATI-2042, tedisamil, Nifekalant, Lu 47110, Ersentilide, trecetilide, almokalant, D-sotalol, BRL-32872, HMR1556, L768673, Vemakalant, AZD70009, AVE0118, S9947, NIP-141/142, XEN-D0101/2, ranolazine, pilsicainide, JTV519, sieve are for adding peptide, GAP-134 or their combination.

In another embodiment, can select for the experimenter who suffers from dysfunction of blood coagulation treatment.One or more biomarkers can be assessed from the vesica from described experimenter, such as, but not limited to F1.2, TAT, FPA, β thromboglobulin, platelet factor 4, solubility CD62P, IL-6 and CRP.According to one or more features of described one or more biomarkers, described experimenter can be defined as to respondent or non-respondent for treatment (such as, but not limited to acetylsalicylic acid, anti-coagulant, ximelagatran, heparin, warfarin or its combination).

In another embodiment, can select for the experimenter who suffers from premature infant's atherosclerosis (Premature Atherosclerosis) treatment.Can assess one or more biomarkers from the vesica from described experimenter, such as, but not limited to CRP, NF-kB, IL-1, IL-6, IL-18, Apo-B, Lp-PLA2, Fibrinogen, Hcy and Hcy-thiolactone.According to one or more features of described one or more biomarkers, described experimenter can be defined as to respondent or non-respondent for treatment.

In another embodiment again, can select treatment for suffering from hypertensive experimenter.Can assess one or more biomarkers from the vesica from described experimenter, such as, but not limited to brain natriuretic peptide and N-terminal prohormone BNP.According to one or more features of described one or more biomarkers, described experimenter can be defined as to respondent or non-respondent for treatment.

In another embodiment, can select for the experimenter who suffers from cardiovascular disorder treatment.Can assess one or more biomarkers from the vesica from described experimenter, such as, but not limited to ACE inhibitor or Angiotensin.According to one or more features of described one or more biomarkers, described experimenter can be defined as to respondent or non-respondent for treatment (such as, but not limited to lisinopril, Candesartan, enalapril or its combination).

Therefore, can be imprinted as according to the biology of experimenter's vesica and suffer from conditions associated or described experimenter cardiovascular status of Cardiology and select treatment.

Autoimmunization

The assessment vesica can be used for the treatment diagnosis of auto immune conditions, imbalance or disease.Auto immune conditions is that mammiferous immunity system starts the situation reacted with himself tissue.These situations include but not limited to, systemic lupus erythematous (SLE), discoid lupus, systemic lupus erythematosus, sarcoidosis, inflammatory arthritis (comprises juvenile arthritis, rheumatoid arthritis, psoriatic arthritis, Reiter syndrome, ankylosing spondylitis and urarthritis), multiple sclerosis, super IgE syndrome, polyarteritis nodosa, the primary biliary cirrhosis liver cirrhosis, inflammatory bowel, Crohn disease, celiac disease (gluten susceptibility enteropathy), autoimmune hepatitis, pernicious anemia, autoimmune hemolytic anemia, psoriasis, scleroderma, myasthenia gravis, autoimmune thrombocytopenic purpura, autoimmune thyroiditis, Graves disease, struma lymphomatosa, immune complex disease, confirmed fatigue immune dysfunction syndrome (CFIDS), polymyositis and dermatomyositis, cryoglobulinemia, thrombolysis, myocardosis, pemphigus vulgaris, interstitial pulmonary fibrosis, asthma, Churg-Strauss syndrome (allergic granuloma), atopic dermatitis, supersensitivity and irritant contact dermatitis, urticaria, IgE mediation type allergy, atherosclerosis, vasculitis, idiopathic inflammatory myositis, hemolytic disease, alzheimer's disease, chronic inflammatory demyelinating polyneuropathy, South American trypanosomiasis, chronic obstructive pulmonary disease, dermatomyositis, type 1 diabetes, endometriosis, Goodpastures syndrome, Graves' disease, guillain-Barre syndrome (gbs), Hashimoto's disease, suppurative hidradenitis, Chuan Qishi disease, IgA nephropathy, idiopathic thrombocytopenic purpura, interstitial cystitis, lupus erythematosus i, mixed connective tissue disease, morphea, myasthenia gravis, narcolepsy, neuromyotonia, pemphigus vulgaris, pernicious anemia, psoriatic, psoriatic arthritis, polymyositis, primary biliary cirrhosis, rheumatoid arthritis, schizophrenia, scleroderma, siogren's syndrome, the stiff man syndrome, temporal arteritis, ulcerative colitis, vasculitis, vitiligo, wegener granulomatosis and AID.

autoimmunization: the biological marking

Can assess the biological marking of vesica and think that the experimenter provides the treatment diagnosis.The biological marking of described vesica can comprise one or more biomarkers, such as, but not limited to, such as in Fig. 1 for autoimmune disease cited biomarker, or the biomarker of other autoimmune disease, such as, but not limited to listed biomarker in Figure 23,34,35,36,39,41,42 and 56.

autoimmunization: nursing standard

The biological marking of the vesica of the sample of the experimenter from suffering from auto immune conditions, imbalance or disease, vesica amount or both are measured and can be used for selecting nursing standard for described experimenter.Most of autoimmune diseases still can not directly be treated, but are treated according to the symptom associated with described situation.Nursing standard comprises, for example, open corticosteroid medication, non-steroidal anti-inflammatory drug (NTHE) or more powerful immunosuppressive drug, such as endoxan, methotrexate and imuran, the progress of disease is replied and stoped to described medicine Immunosuppression.To the irradiation of lymphoglandula and plasma exchange, (measure that diseased cells and harmful molecule are removed from blood circulation) is the alternate manner for the treatment of autoimmune disease.

The medicine that is used for the treatment of autoimmune disease that the somatotype of biomarker that can be based on from the experimenter is selected or the example of medicament comprise in table 13 medicine of the experimenter for suffering from diabetes, in table 14 for suffering from the medicine of multiple sclerosis.

Table 13: for the example of the drug categories for the treatment of diabetes

Table 14: for the drug categories of multiple sclerosis therapy

In one embodiment, the detection of the miR-326 from vesica be can be used for characterizing multiple sclerosis, and can be one or more treatments that described experimenter is selected from table 14.In another embodiment, described sign can comprise the therapy of selecting such as interferon beta-1b and interferon beta-1a.

In another embodiment, can select for the experimenter who suffers from rheumatoid arthritis treatment.Can assess one or more biomarkers from the vesica from described experimenter, such as, but not limited to 677CC/1298AA MTHFR, 677CT/1298AC MTHFR, 677CT MTHFR, G80AA RFC-1, 3435TT MDR1 (ABCB1), 3435TT ABCB1, AMPD1/ATIC/ITPA, IL1-RN3, HLA-DRB103, CRP, HLA-D4, HLA DRB-1, containing anti-citrulline epitope peptide, anti-A1/RA33, erythrocyte sedimentation rate (ESR), c reactive protein (CRP), SAA (serum amyloid sample associated protein), Rheumatoid factors, polyclonal, IL-1, TNF, IL-6, IL-8, IL-1Ra, hyaluronic acid, aggrecan, Glc-Gal-PYD, osteoprotegerin, RNAKL, cartilage oligo-substrate protein (COMP) and calprotectin.According to one or more features of described one or more biomarkers, described experimenter can be defined as to reactor or the non-reactor for the treatment of (such as, but not limited to methotrexate, infliximab, adalimumab, etanercept, sulfasalazine or its combination).

Therefore, can be imprinted as the experimenter who suffers from auto immune conditions according to the biology of experimenter's vesica and select treatment.

Infectious diseases

Assessment to vesica can be used for treating the diagnose infections disease, such as bacillary, viral or other infectious situation or disease.Infectivity or parasitosis can come from bacterium, virus, fungi or other parasitic infection.For example, described disease or situation can be Whipples disease, prion disease, liver cirrhosis, methicillin-resistant staphylococcus aureus, HIV, hepatitis, syphilis, meningitis, malaria, tuberculosis or influenza.

Infectivity or parasitic disease can include but not limited to that intestines catch, tuberculosis, zoogenous bacteriosis, other bacteriosis, human immunodeficiency virus hiv infects, poliomyelitis and other non-arthropod-borne central nervous system virus disease, the virus disease that fash is followed, arthropod-borne virus disease, due to virus and the Other diseases that causes of chlamydozoan, Dermacentroxenus and other arthropod-borne disease, syphilis and other venereal disease, other spirochete disease, mycosis, verminosis, other infectivity and parasitic disease, and the anaphase effect of infectious and parasitic disease.The intestines infectious diseases includes but not limited to cholera, Typhoid and paratyphoid, Salmonellas gastro-enteritis, shigellosis, not clear shigellosis, Staphylococcus food poisoning, loeschiasis, acute amebic dysentery (not addressing abscess), chronic enteron aisle loeschiasis (not addressing abscess), non-dysentery amebic colitis, amebic liver abscess, amebic abscess of lung, cerebral amebic abscess, the amebic skin ulcer, the amoeba infection at other position, not clear loeschiasis, balantidiasis, giardiasis, coccidiosis, intestinal trichomoniasis, cryptosporidiosis, Cyclosporiasis, not clear protozoon property enteropathy, by the intestinal tract infections due to other biology, due to the enteritis due to rotavirus, by the enteritis due to other viral enteritis, that does not classify in addition is infected by the intestines due to other organism, indefinite intestines infect, the gastro-enteritis of colitis enteritis and the supposition source of infection.

The human immunodeficiency virus infection includes but not limited to, the human immunodeficiency virus infection of the clear and definite situation of tool, the human immunodeficiency virus infection who causes other clear and definite situation and other human immunodeficiency virus infection.

Poliomyelitis and other non-arthropod-borne central nervous system virus disease include but not limited to, the slow virus infection of acute poliomyelitis, central nervous system, kuru (kuru), creutzfeldt-Jacob disease, the meningitis due to due to enterovirus, other enterovirus disease and other non-arthropod-borne central nervous system virus disease of central nervous system.The virus disease that fash is followed includes but not limited to smallpox, cowpox and paravaccinia, varicella, zoster, herpes simplex, genital herpes, herpetic gingivostomatitis, uncomplicated herpetic disease, measles, rubella, other viral exanthemata, No. five diseases, not clear viral fash, roseola infantum, other herpes virus hominis's property encephalitis, other herpes virus hominis infects, other poxvirus infection, other poxvirus infection, monkeypox, other parapoxvirus infects, the ox stomatitis, the sea dog acne, inferior tower poxvirus infection, Tanapox, yaba monkey tumor virus, other poxvirus infection and not clear poxvirus infection.

Arthropod-borne virus disease includes but not limited to, yellow jack, singapore hemorrhagic fever, carapuru virus encephalitis, the not clear viral encephalitis of mosquito matchmaker, tickborne virus encephalitis, other and not clear arthropod-borne viral encephalitis, arthropod-borne hemorrhagic fever, not clear ebola disease, other arthropod-borne virus disease and not clear west Nile virus.

Because the Other diseases due to virus and chlamydozoan includes but not limited to viral hepatitis, the hepatitis A hepatic coma that occurs together, hepatitis A without stupor, the hepatitis B hepatic coma that occurs together, hepatitis B without stupor, the viral hepatitis (addressing hepatic coma) that acute other is clear and definite, other clear and definite viral hepatitis (not addressing hepatic coma), indefinite viral hepatitis C, viral hepatitis C's hepatic coma that occurs together, viral hepatitis C's hepatic coma that occurs together, viral hepatitis, rabies, parotitis, uncomplicated parotitis, ornithosis, due to the clear and definite disease due to Coxsackie virus, herpangina, hand foot mouth disease, mononucleosis, trachoma, due to virus and chlamydozoan due to other conjunctivitis disease, due to virus and chlamydozoan due to Other diseases, the contact molluscum, the verrucosis of all sites, pointed condyloma, perspire and generate heat, cat scratch disease, pin and stomatosis, the CMV disease, virus infection in the situation of classifying in addition and not clear position, rhinovirus, hpv and respiratory syncytial virus.Rickettsiae and other arthropod-borne disease include but not limited to, disease, Lyme disease and the Babesia disease of louse-borne epidemic typhus, other typhus fever, tick matchmaker rickettsiosis, Rocky Mountain spotted fever, other rickettsiosis, malaria, leishmaniasis, trypanosomiasis (trypa omiasis), typhinia, other arthropod-borne disease, other clear and definite arthropod-borne infection.

Virus host includes but not limited to adenovirus, Astrovirus, avian influenza virus, Coxsackie virus, dengue fever virus, Ebola virus, Echo virus, EAd, enterovirus, Hantaan virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, hsv (HSV), human cytomegalic inclusion disease virus, human immunodeficiency virus (HIV), human papillomavirus (HPV), influenza virus, japanese encephalitis virus (JEV), Lassa fever virus, Marburg virus, Measles virus, mumps virus, Norwalk virus, parainfluenza virus, poliovirus, rabies virus, respiratory syncytial virus, rotavirus, rubella virus, sars coronavirus, Far East Russian encephalitis virus (TBEV), variola virus, west Nile virus and yellow fever virus.Fungal host includes but not limited to Candida albicans.Parasite the host include but not limited to, plasmodium, Schistosoma mansoni and Trichomonas vaginalis.

Host bacterium includes but not limited to, Acinetobacter baumannii, Bacillus anthracis, Bartonella, bordetella pertussis, the uncle Bordetella, Brucella, Chlamydia pneumoniae, chlamydia trachomatis, Clostridium botulinum, corynebacterium diphtheriae, Coxiella burnetii, Ehrlichia, enterococcus spp, enterovirus, intestinal bacteria, soil draws not Salmonella, Haemophilus ducreyi, helicobacter pylori, Klebsiella pneumonia, legionella pneumophilia, leptospira interrogans, mycobacterium tuberculosis, mycoplasma genitalium, Chlamydia pneumoniae, Diplococcus gonorrhoeae, Neisseria meningitidis, Orientia Tsutsugamushi (orientia tsutsugamushi), Pseudomonas aeruginosa, Rickettsiae, Salmonellas, Shigellae, streptococcus aureus, streptococcus pneumoniae, micrococcus scarlatinae, Tyreponema pallidum, ureaplasma urealyticum, vibrio cholerae, Vibrio vulnificus and Yersinia pestis.

Zoogenous bacteriosis includes but not limited to, pestilence, glandular plague, tularemia, anthrax, brucellosis, glanders, pseudoglanders, large rat-bite fever, listeriosis, erysipelothrix infection and Bacillus pasteurii disease.Other bacteriosis includes but not limited to leprosy, due to other mycobacterium associated diseases, diphtheria, Whooping cough, suis is had a sore throat and scarlet fever, suis pharyngitis, scarlet fever, erysipelas, meningococcal meningitis, tetanus, septicemia, pneumococcal septicemia, gram-negative sepsis, not clear septicemia and actinomycotic infection.

Tuberculosis includes but not limited to, follows erythema nodosum, bazin disease (bazin disease), peripheral lymph nodes tuberculosis, tuberculous lymphadenitis and the granular tuberculosis of grain of allergy in primary tuberculous infection, pulmonary tuberculosis, meninx and tuberculosis of central nervous system, intestines, peritonaeum and tuberculosis of mesentric lymph nodes disease, bone and joint tuberculosis, spinal tuberculosis, pott's disease (pott ' s disease), tuberculosis of genitourinary system disease, other organ tuberculosis, tuberculosis.

Syphilis and other venereal disease include but not limited to congenital syphilis, tool symptom early syphilis, primary genitalia syphilis, latent early syphilis, cardiovascular syphilis, neurosyphilis, the tool symptom tertiary syphilis of other form, the tertiary syphilis of hiding, other and indefinite syphilis, gonococcal infection, acute gonorrhea of lower genitourinary tract, gonococcal conjunctivitis and nongonococcal urethritis.Other spirochete disease includes but not limited to leptospirosis, ulceromembranous angina, yaws and pinta.Mycosis includes but not limited to tinea, tinea pedis, ringworm of the body, other and not clear dermatophytes property, the tinea versicolor of beriberi, scalp/beard beriberi, tinea unguium, arm, not clear tinea, moniliosis, oral candidiasis, vulva/vaginal candidiasis, candidal balanitis, skin/refer to toenail moniliosis, coccidioidomycosis, histoplasmosis, the infection of not clear histoplasma capsulatum, blastomycotic infection, other mycosis and opportunistic mycosis.

Verminosis includes but not limited to, schistosomicide, other fluke infection, hydatidosis, other cestode infection, trichi is, filarial infection and dracunculiasis, uncinariasis and necatorosis, other intestinal helminthiasis, ascariasis, anisakiasis, strongyloidiasis, trichuriasis, oxyuriasis, capillariasis, trichostrongyliasis, other and not clear verminosis and not clear intestinal parasitical diseases.Other infectious and or parasitosis include but not limited to toxoplasmosis, not clear toxoplasmosis, trichomoniasis, urogenital tract trichomoniasis, trichomonal vaginitis, trichomonad urethritis, pediculosis and crab louse invasions, head louse pediculosis, body louse pediculosis, crab louse pediculosis, fail to understand pediculosis, scabies, mange, trombiculid, sarcoidosis, ainhum, Behcet's syndrome, pneumocystosis, psorospermisis and sarcosporidiasis.Anaphase effect infectious and parasitic disease includes but not limited to anaphase effect lungy and poliomyelitic anaphase effect.

infectious diseases: the biological marking

Can assess the biological marking of vesica and think that the experimenter provides the treatment diagnosis.The biological marking of described vesica can comprise one or more biomarkers, such as, but not limited to, any one or multiple biomarker as herein described, as but be not limited to, in the biomarker of listing for infectious diseases in Fig. 1 and Figure 24 and 43, list biomarker.

In some embodiments, can characterize infectious diseases by the composition that detects the pathogenic agent (such as virus, bacterium or other infectious agent) in vesica.For example, described composition can be abc transport body (Candida albicans), abc transport body (enterococcus), AMA-1 (teleblem antigen 1), the ATP enzyme, Aac (6 ')-Aph (2 ") enzyme, Ace (pasracholera enterotoxin), Acf (the auxiliary factor of surely growing), Acr (alpha-crystal albumen) albumen, AhpC and AhpD, amyloid-beta, AroC, adhere to glycoprotein (G) (Respiratory Syncytial Virus(RSV)), autolysin (N-acetyl muramyl-ALANINE amidase), BacA, BmpA (P39), botulic neurotoxin, BvgA ,-S and-R, BvrR-BvrS, C4BP (C4b is in conjunction with albumen), the C5a peptase, CAMP factor (haemolysis promotes the factor (cohemolysin)), CBP (choline binding protein), CME type beta-lactamase, CSP (circumsporozoite protein), CT (cholera toxin), the CTX-M metal-beta-lactamase, CagA (cytotoxin related antigen), capsid protein (C) (dengue fever virus), capsid protein (C) (japanese encephalitis virus), capsid protein (C) (tick borne encephalitis virus), capsid protein (C) (West Nile Virus), capsid protein (C) (flavivirus), capsid protein (astrovirus), capsid protein (Coxsackie virus), capsid protein (echovirus), capsid protein (enterovirus), capsid protein (hepatitis A virus), capsid protein (poliovirus), capsid protein (rotavirus), catechol siderophore abc transport body, Com-1, CrmB (cell factor reaction control agent), cytolysin, the D-Ala-D-Lac ligase, DHFR (dihyrofolate reductase), DHPS (Dihydropetorate synthase), DbpA (decorin is in conjunction with albumin A), diphtheria toxin, the Dot/Icm complex, E1 and E2 albumen (rubella virus), E1A albumen (adenovirus), E1A albumen (EAd), E1B albumen (adenovirus), E1B albumen (EAd), E2 early transcription district 2, E3 albumen (adenovirus), E4 albumen (adenovirus), E6 early transcription district 6, E7 early transcription district 7, EF (edema factor), ESAT-6 and CFP-10, elastoser (Vibrio vulnificus), Env, envelope glycoprotein (E) (dengue fever virus), envelope glycoprotein (E) (japanese encephalitis virus), envelope glycoprotein (E) (tick borne encephalitis virus), envelope glycoprotein (E) (West Nile Virus), envelope glycoprotein (E) (flavivirus), Esp (surface protein), Esp (III type system secretion protein), F1 tunicle (F1 antigen), FH (factor H), FHA (filamentous hemagglutinin), Falcipain 1/2, fibrin (adenovirus), fibrin (EAd), fibronectin binding protein II (albumen F/sfbII) (micrococcus scarlatinae), fibronectin binding protein (leptospira interrogans), fibronectin binding protein (FBP54) (micrococcus scarlatinae), dynein, flagellin (FlaB and-A) (helicobacter pylori), flagellin (H-antigen) (Escherichia coli), flagellin (H-antigen) (salmonella), flagellin (Vibrio vulnificus), FopA (43kDa lipoprotein), fusion (F) (mumps virus), fusion (F) (parainfluenza virus), fusion (F) (Respiratory Syncytial Virus(RSV)), G6PD (glucose-6-phosphate dehydrogenase (G6PD)), GES (Guyana extended spectrumβ-lactamase), the GTP cyclohydrolase, Gag, glycoprotein (G) (hydrophobin), glycoprotein (GP) (Ebola virus), glycoprotein (GP) (Lassa virus), glycoprotein (GP) (Marburg virus), glycoprotein (Gn/Gn) (Hantaan virus), HMW (Cytadherence auxilin), HRP2 (histidine rich protein 2), hemagglutinin (avian influenza virus), hemagglutinin (influenza virus), hemagglutinin (measles virus), hemagglutinin (variola virus), hemagglutinin esterase glycoprotein (HE), hemagglutinin-neuraminidase (HN) (mumps virus), hemagglutinin-neuraminidase (HN) (parainfluenza virus), hemolysin (Vvh), hexon (adenovirus), hexon (EAd), HSP60 (HSP60), hyaluronate lyase, hyaluronidase, IMP metal-beta-lactamase (Acinetobacter baumannii), IMP metal-beta-lactamase (Klebsiella Pneumoniae), ICSA and ICSB, IgA protease (gonococcus), IgA1 protease (streptococcus pneumonia), the IgG of HSV 1/2 and IgM, InhA, Intimin, InvA (rickettsia), invasion (Escherichia coli), invasion (Yersinia pestis), IpaA,-B,-C,-D and-H, the KPC metal-beta-lactamase, KatG, L albumen (Lassa virus), L1 late transcription district 1, LF (lethal factor), LSA1 (liver stage antigens 1), LT (heat-labile toxin), LcrV (V antigen), LigA and LigB, lipoprotein, M albumen, MSP (merozoite surface protein), stromatin (M) (hydrophobin), stromatin (M) (Respiratory Syncytial Virus(RSV)), stromatin (avian influenza virus), stromatin (influenza virus), MexAB-OprM, MexCD-OprJ, MexEF-OprN, MexXY-OprM, Mip (the infectious reinforcing agent of macrophage), NSE (neuron specific enolase), Nef, neuraminidase (avian influenza virus), neuraminidase (influenza virus), neuraminidase (streptococcus pneumonia), non-structural protein (NS) (Respiratory Syncytial Virus(RSV)), non-structural protein 1 (NS1) (dengue fever virus), non-structural protein 1 (NS1) (japanese encephalitis virus), non-structural protein 1 (NS1) (tick borne encephalitis virus), non-structural protein 1 (NS1) (West Nile Virus), non-structural protein 1 (NS1) (flavivirus), non-structural protein 2A (NS2A) (dengue fever virus), non-structural protein 2A (NS2A) (japanese encephalitis virus), non-structural protein 2A (NS2A) (tick borne encephalitis virus), non-structural protein 2A (NS2A) (West Nile Virus), non-structural protein 2A (NS2A) (flavivirus), non-structural protein 2B (NS2B) (dengue fever virus), non-structural protein 2B (NS2B) (japanese encephalitis virus), non-structural protein 2B (NS2B) (tick borne encephalitis virus), non-structural protein 2B (NS2B) (West Nile Virus), non-structural protein 2B (NS2B) (flavivirus), non-structural protein 3 (NS3) (dengue fever virus), non-structural protein 3 (NS3) (japanese encephalitis virus), non-structural protein 3 (NS3) (tick borne encephalitis virus), non-structural protein 3 (NS3) (West Nile Virus), non-structural protein 3 (NS3) (flavivirus), non-structural protein 4 (rotavirus), non-structural protein 4A (NS4A) (dengue fever virus), non-structural protein 4A (NS4A) (japanese encephalitis virus), non-structural protein 4A (NS4A) (tick borne encephalitis virus), non-structural protein 4A (NS4A) (West Nile Virus), non-structural protein 4A (NS4A) (flavivirus), non-structural protein 4B (NS4B) (dengue fever virus), non-structural protein 4B (NS4B) (japanese encephalitis virus), non-structural protein 4B (NS4B) (tick borne encephalitis virus), non-structural protein 4B (NS4B) (West Nile Virus), non-structural protein 4B (NS4B) (flavivirus), Non structural protein 5 (NS5) (dengue fever virus), Non structural protein 5 (NS5) (japanese encephalitis virus), Non structural protein 5 (NS5) (tick borne encephalitis virus), Non structural protein 5 (NS5) (West Nile Virus), Non structural protein 5 (NS5) (flavivirus), non-structural protein (avian influenza virus), non-structural protein (influenza virus), nucleocapsid (Hantaan virus), nucleocapsid (measles virus), nucleocapsid (parainfluenza virus), nucleocapsid (sars coronavirus), nucleoprotein (N) (hydrophobin), nucleoprotein (NP) (Respiratory Syncytial Virus(RSV)), nucleoprotein (main nucleoprotein) (Marburg virus), nucleoprotein (avian influenza virus), nucleoprotein (Ebola virus), nucleoprotein (influenza virus), nucleoprotein (Lassa virus), ORF1 (HEV), ORF2 (HEV), ORF3 (HEV), OXA metal-beta-lactamase (Acinetobacter baumannii), OXA metal-beta-lactamase (Klebsiella Pneumoniae), OmpA and OmpB (rickettsia), OmpL1 (leptospira interrogans), OmpQ (adventitia duct albumen) (Bordetella pertussis), OmpS (legionella pneumophilia), turbidity factor, OprD, Osp (outer surface protein), outer membrane protein (CPN), outer membrane protein (Ehrlichia), the P1 adhesin, the P30 adhesin, PA (protective antigens), PBP (PBP), PCRMP 1-4 (cysteine replicated blocks protein), the PER metal-beta-lactamase, Pat1, peptide glycan (murein) hydrolase, pertactin (P69), pertussis toxin, PfEMP1 (plasmodium falciparum erythrocyte membrane protein 1), phosphoprotein (P) (Respiratory Syncytial Virus(RSV)), phosphoprotein (measles virus), Pla (PLA), plasminogen is in conjunction with albumen, Pld, pneumolysin, Pol, poly--D-Glu coating, polymerase (L) (rabies viruses), duct albumen, cephacoria/memebrane protein (PRM/M) (dengue fever virus), cephacoria/memebrane protein (PRM/M) (japanese encephalitis virus), cephacoria/memebrane protein (PRM/M) (tick borne encephalitis virus), cephacoria/memebrane protein (PRM/M) (West Nile Virus), cephacoria/memebrane protein (PRM/M) (flavivirus), the albumen of bi-component regulating system (Ehrlichia), the albumen of bi-component regulating system (Much's bacillus), gB, gC, gD, gH and gL albumen, PsaA, PspA (Pneumococal surface protein A), PurE, the pyrogen exotoxin, RBP 1/2 (granulophilocyte Binding Protein 1/2), RdRp (RNA RNA-dependent polymerase) (norovirus), RdRp (RNA RNA-dependent polymerase) (astrovirus), RdRp (RNA RNA-dependent polymerase) (sars coronavirus), Rev, RfbE, RibD and RibE, Rmp, S-layer albumen, S100B (S100 albumen β chain), the SHV metal-beta-lactamase, the SIM metal-beta-lactamase, ST (heat-stable toxin), detection of Salmonella plasmid virulence (SPV) albumen, serine protease (astrovirus), ShET1/2, shiga toxin (syphilis virus), SipA (salmonella invasin protein A), SlyA, little hydrophobic proteins, Sop (the outer albumen of salmonella), spike glycoprotein (S), streptodornase, the Streptogramin A transacetylase, streptokinase, streptolysin O, StxA/B (shiga toxin A/B), SucB (dihydrolipoamide succinyltransferase) (Much's bacillus), SucB (dihydrolipoamide succinyltransferase) (Coxiella burnetii), Syc (Yop molecular chaperones), T albumen, TCP (toxin-coregulated pili), the TEM metal-beta-lactamase, TRAP (thrombostondin be correlated with unnamed protein), Tat, Tau albumen, TcfA (tracheae is grown the factor surely), Tir (transposition intimin acceptor), TlyA and TlyC, ToxR (toxin adjusting albumen), Tul4 (17kDa lipoprotein), IV type pili, urase (brucella), urase (helicobacter pylori), the VEB metal-beta-lactamase, VETF (the viral early transcription factor), VIM metal-beta-lactamase (Acinetobacter baumannii), VIM metal-beta-lactamase (Klebsiella Pneumoniae), VP1 (norovirus), VP2 (norovirus), VP24 (Ebola virus), VP24 (Marburg virus), VP30 (small nucleoprotein) (Ebola virus), VP30 (small nucleoprotein) (Marburg virus), VP35 (P-sample protein) (Ebola virus), VP35 (P-sample protein) (Marburg virus), VP40 (stromatin) (Ebola virus), VP40 (stromatin) (Marburg virus), VacA (physalis toxin), Vag8 (virulence activated gene 8), Vif albumen, VirB IV type excretory system, VlsE (35kDa lipoprotein), Vpr, Vpu/Vpx, XerD, Yops (Yersinia ruckeri outer membrane protein), Ysc (Yop secernent), Z albumen (Lassa virus), Zot (zonuls occludens toxin), GG1 (HSV-1) and GG2 (HSV-2), p41i, p83 and p100, pLDH (pLDH), α/β/γ albumen, the 120kDa gene, 16S and 5SrRNA gene (legionella pneumophilia), 16S rRNA gene (Bartonella), 16S rRNA gene (Borrelia), 16S rRNA gene (brucella), 16S rRNA gene (Ehrlichia), 16S rRNA gene (Klebsiella Pneumoniae), 16S rRNA (Orientia Tsutsugamushi), 16S rRNA gene (rickettsia), 16S rRNA gene (Acinetobacter baumannii), 16S rRNA gene (CPN), 16S rRNA gene (clostridium botulinum), 16S rRNA gene (mycoplasma pneumoniae), 16S rRNA gene (Diplococcus gonorrhoeae), 16S rRNA gene (Vibrio vulnificus), 16S-23S rRNA intergenic region (Bartonella), 16S-23S rRNA intergenic region (Coxiella burnetii), the 17kDa gene, 18S ssrRNA, 23S rRNA gene (Acinetobacter baumannii), 23S rRNA gene (Diplococcus gonorrhoeae), the 2C gene, 3 ' NCR (dengue fever virus), 3 ' NCR (japanese encephalitis virus), 3 ' NCR (tick borne encephalitis virus), 3 ' NCR (West Nile Virus), 3 ' NCR (flavivirus), 5 ' NCR (Coxsackie virus), 5 ' NCR (dengue fever virus), 5 ' NCR (echovirus), 5 ' NCR (enterovirus), 5 ' NCR (japanese encephalitis virus), 5 ' NCR (poliovirus), 5 ' NCR (tick borne encephalitis virus), 5 ' NCR (West Nile Virus), 5 ' NCR (flavivirus), the 56kDa gene, the A13L gene, the ARE1 gene, the ATF2 gene, the B12R gene, the B6R gene, the B8R gene, C gene (dengue fever virus), C gene (japanese encephalitis virus), C gene (tick borne encephalitis virus), C gene (West Nile Virus), C gene (flavivirus), the C3L gene, CDR 1/2 gene, E gene (dengue fever virus), E gene (japanese encephalitis virus), E gene (tick borne encephalitis virus), E gene (West Nile Virus), E gene (flavivirus), E1 and raq gene, e1a gene (adenovirus), e1a gene (EAd), E1B gene (adenovirus), E1B gene (EAd), raq gene, E3 gene (adenovirus), the E3L gene, E4 gene (adenovirus), the E6 gene, the E7 gene, the ERG gene, ESAT-6 and CFP-10 gene, F gene (mumps virus), F gene (parainfluenza virus), F gene (Respiratory Syncytial Virus(RSV)), G gene (hydrophobin), G gene (Respiratory Syncytial Virus(RSV)), GP gene (Ebola virus), GP gene (Lassa virus), GP gene (Marburg virus), H gene (measles virus), HA gene (avian influenza virus), HA gene (influenza virus), HE gene (sars coronavirus), HN gene (mumps virus), HN gene (parainfluenza virus), IS100, IS1081, IS1533 (leptospira interrogans), IS285, IS481 (BP0023), IS6110, IS711 (brucella), ISFtu, the J7R gene, L gene (Lassa virus), L gene (hydrophobin), the L fragment, the L1 gene, LEE (locus that enterocyte is eliminated), LCR (LCR), M gene (hydrophobin), M gene (Respiratory Syncytial Virus(RSV)), M gene (avian influenza virus), M gene (influenza virus), the M fragment, the MDR1 gene, the MEC3 gene, N gene (measles virus), N gene (hydrophobin), N gene (sars coronavirus), NA gene (avian influenza virus), NA gene (influenza virus), NC gene (parainfluenza virus gene), NP gene (avian influenza virus), NP gene (Ebola virus), NP gene (influenza virus), NP gene (Lassa virus), NP gene (Marburg virus), NP gene (Respiratory Syncytial Virus(RSV)), NS gene (avian influenza virus), NS gene (influenza virus), NS gene (Respiratory Syncytial Virus(RSV)), NS1 gene (dengue fever virus), NS1 gene (japanese encephalitis virus), NS1 gene (tick borne encephalitis virus), NS1 gene (West Nile Virus), NS1 gene (flavivirus), NS2A gene (dengue fever virus), NS2A gene (japanese encephalitis virus), NS2A gene (tick borne encephalitis virus), NS2A gene (West Nile Virus), NS2A gene (flavivirus), NS2B gene (dengue fever virus), NS2B gene (japanese encephalitis virus), NS2B gene (tick borne encephalitis virus), NS2B gene (West Nile Virus), NS2B gene (flavivirus), NS3 gene (dengue fever virus), NS3 gene (japanese encephalitis virus), NS3 gene (tick borne encephalitis virus), NS3 gene (West Nile Virus), NS3 gene (flavivirus), NS4 gene (rotavirus), NS4A gene (dengue fever virus), NS4A gene (japanese encephalitis virus), NS4A gene (tick borne encephalitis virus), NS4A gene (West Nile Virus), NS4A gene (flavivirus), NS4B gene (dengue fever virus), NS4B gene (japanese encephalitis virus), NS4B gene (tick borne encephalitis virus), NS4B gene (West Nile Virus), NS4B gene (flavivirus), NS5 gene (dengue fever virus), NS5 gene (japanese encephalitis virus), NS5 gene (tick borne encephalitis virus), NS5 gene (West Nile Virus), NS5 gene (flavivirus), ORF 1A (astrovirus), ORF 1B (astrovirus), ORF 2 (astrovirus), ORF1 (HEV), ORF1 (norovirus), ORF2 (HEV), ORF2 (norovirus), ORF3 (HEV), ORF3 (norovirus), P gene (measles virus), P gene (Respiratory Syncytial Virus(RSV)), the PDH1 gene, the transpeptidation enzyme mutant, plasmid (QpH1, QpRS, QpDG, QpDV), PrM/M gene (dengue fever virus), PrM/M gene (japanese encephalitis virus), PrM/M gene (tick borne encephalitis virus), PrM/M gene (West Nile Virus), PrM/M gene (flavivirus), RdRp gene (sars coronavirus) in ORF 1ab, S gene (sars coronavirus), the S fragment, SH gene (mumps virus), SNP (SNP), salmonella pathogenicity island (SPI), salmonella plasmid virulence (SPV) operon, the ShET1/2 gene, VNTR (variable number tandem repeat) (bacillus anthracis), VNTR (variable number tandem repeat) (brucella), VNTR (variable number tandem repeat) (Francisella tularensis), VNTR (variable number tandem repeat) (Yersinia pestis), VP24 gene (Ebola virus), VP24 gene (Marburg virus), VP30 gene (Ebola virus), VP30 gene (Marburg virus), VP35 gene (Ebola virus), VP35 gene (Marburg virus), VP40 gene (Ebola virus), VP40 gene (Marburg virus), Z gene (Lassa virus), aac (3) gene, aac (6 ') gene, aac (6 ')-aph (2 ") gene, the aad gene, the ace gene, the acpA gene, the agrBDCA locus, ahpC and ahpD gene, the arlRS locus, the atxA gene, the bclA gene, the blaCTX-M gene, the blaGES gene, blaGIM gene (Pseudomonas aeruginosa), blaIMP gene (Acinetobacter baumannii), blaIMP gene (Klebsiella Pneumoniae), blaIMP gene (Pseudomonas aeruginosa), the blaKPC gene, blaOXA gene (Acinetobacter baumannii), blaOXA gene (Klebsiella Pneumoniae), blaOXA gene (Pseudomonas aeruginosa), the blaSHV gene, blaSIM gene (Klebsiella Pneumoniae), blaSIM gene (Pseudomonas aeruginosa), the blaTEM gene, blaVIM gene (Acinetobacter baumannii), blaVIM gene (Klebsiella Pneumoniae), blaVIM gene (Pseudomonas aeruginosa), bvg locus (bvgA,-S and-the R gene), the cagA gene, cap locus (capB,-C and-the A gene) (bacillus anthracis), cap operon (capB and-C) (Francisella tularensis), capsid gene (Coxsackie virus), capsid gene (echovirus), capsid gene (enterovirus), capsid gene (hepatitis A virus), capsid gene (poliovirus), capsid gene (rotavirus), the cme gene, the cnt gene, the com-1 gene, the cppB gene, the cps gene, the crmB gene, the ctx gene, the cya gene, the cyl gene, the eaeA gene, east gene (Escherichia coli), the env gene, the ery gene, esp gene (enterococcus), esp gene (Escherichia coli), fiber gene (adenovirus), fiber gene (EAd), fimbriae gene, flaB gene (Borrelia), flaB gene (leptospira interrogans), flagellin gene, fljA, fljB and fliC gene, the fopA gene, the ftsZ gene, gG1 and gG2 gene, the gag gene, the gene of bi-component regulator control system, gB, gC, gD, the gene of gH and gL, gerX locus (gerXC,-A and-the B gene), the glpQ gene, gltA (citrate synthetase) gene (Bartonella), gltA (citrate synthetase) gene (rickettsia), groEL gene (Bartonella), groEL gene (Orientia Tsutsugamushi), groESL gene (CPN), gyrA and gyrB gene (Pseudomonas aeruginosa), gyrA gene (Diplococcus gonorrhoeae), gyrB gene (bacillus anthracis), hexon gene (adenovirus), hexon gene (EAd), the hin gene, the hlyA gene, the hmw gene, hspX (Rv2031c) gene, the htpAB repeat element (IS1111a) of being correlated with, the hyl gene, icsA and icsB gene, the ileS gene, the inhA gene, inv gene (Escherichia coli), inv gene (salmonella), ipaA,-B,-C,-D and-the H gene, the katG gene, the lef gene, the letA gene, the lidA gene, the lpsB gene, the lrgAB locus, the luxS gene, the lytA gene, the lytRS locus, the mecA gene, the mglA gene, mgrA (rat) gene, the mip gene, the mtgA gene, the mucZ gene, multigene family, the mupA gene, nanA and nanB gene, the nef gene, omp gene (brucella), omp gene (CPN), ompA and B gene (rickettsia), the ompQ gene, the opa gene, the osp gene, the p1 gene, the p30 gene, the pagA gene, the pap31 gene, parC and parE gene (Pseudomonas aeruginosa), parC gene (Diplococcus gonorrhoeae), the per gene, the pilQ gene, the ply gene, the pmm gene, the pol gene, porA and porB gene, prn4 (pertactin) gene, the psaA gene, the pspA gene, pst1 fragment and HL-1/HR-1 primer, ptx (promoter region and complete genome), rap 1/2 gene, the rev gene, the rpo18 gene, the rpoB gene, the rpoS gene, the rpsL gene, rrf (5S)-rrL (23S) intergenic region, the rek gene, rtx gene (Vibrio vulnificus), rtxA gene (legionella pneumophilia), sap gene (bacillus anthracis), the sar gene, satA (vatD) and satG (vatE) gene, the sca4 gene, the secY gene, stx (vt) gene, stxA/B (stx1/2) gene, the sucB gene, the tat gene, the tcp gene, the tir gene, the tox gene, the toxR gene, the tul4 gene, the urase gene, the vacA gene, van A-E gene, the veb gene, the vif gene, the viuB gene, the vpr gene, the vpu/vpx gene, vvh (Vibrio vulnificus hemolysin) gene, vvpE (Vibrio vulnificus elastoser) gene, the wboA gene, wzy (O-antigen polymerase) gene, the zot gene, α/β/γ gene, C-polysaccharide (rhamnose/N-acetyl-glucosamine), CPS (coating polysaccharide), ring-type β-1,2 glucan, hyaluronic acid shell coating, LPS (lipopolysaccharides) (Bartonella), LPS (lipopolysaccharides) (brucella), LPS (lipopolysaccharides) (Coxiella burnetii), LPS (lipopolysaccharides) (rickettsia), LPS (lipopolysaccharides) (Vibrio vulnificus), O-antigen (Escherichia coli), O-antigen (salmonella), O-antigen (comma bacillus), Vi-antigen (salmonella) or catechol siderophore.

infectious diseases: nursing standard

The biological marking of the vesica of the sample of the experimenter from suffering from infectivity or parasitic disease, vesica amount or both are measured and can be used for selecting nursing standard for described experimenter.Infectivity or parasitic disease can be treated according to the symptom relevant to described situation.Described nursing standard comprises, for example, uses one or more microbiotic and antiviral agent to be treated.

Microbiotic includes but not limited to amikacin, gentamicin, kantlex, Liu Suanyan NEOMYCIN SULPHATE, netilmicin, Streptomycin sulphate, tobramycin, paromycin, geldanamycin, herbimycin, loracarbff, ertapenem, S-4661, imipenum/cilastatin, meropenem, S 578, Kefzol, cefoxitin or Reflin, Cephalexin Monohydrate Micro/Compacted, cefaclor, Cefamandole, cefoxitin, Prozef, cephalofruxin, Cefixime Micronized, Cefdinir, cefditoren, cefoperazone, cefotaxime, Cefpodoxime, ceftazime, Ceftibuten, ceftizoxime, ceftriaxone, cefepime, Ceftobiprole, teicoplanin, vancomycin, Azythromycin, clarithromycin, dirithromycin, erythromycin, Roxithromycin, troleomycin, Ketek, spectinomycin, aztreonam, amoxycilline Trihydrate bp, penbritin, azlocillin, Pyocianil, Cloxacillin Sodium, dicloxacillin, Flucloxacillin, mezlocillin, X-1497, nafcillin, Oxazacillin, penicillin, piperacillin, ticarcillin, bacitracin, polymyxin, PXB, Ciprofloxacin, enoxacin, Gatifloxacin, levofloxacin, lomefloxacin, Moxifloxacin, norfloxicin, Ofloxacine USP 23, trovafloxacin, grepafloxacin, Sparfloxacin, temafloxacin, mafenide, prontosil (Sulfonamidochrysoidine), sulfacetamide, sulfanilic amide, sulfasalazine, Sulfafurazole, trimethoprim, trimethoprim, Sulfamethoxazole, Demethylchlortetracycline, Vibravenos, Minocycline HCl, terramycin, tsiklomitsin, Sulphadiazine Sodium, sulfamethylthiadiazole, Arsphenamine, paraxin, clindamycin, lincomycin, Tibutol, phosphonomycin, fusidic acid, Nifurazolidone, vazadrine, profit is azoles how, metronidazole, mupirocin, furadantin, dull and stereotyped mycin, pyrazinoic acid amide, Quinupristin or dalfopristin, Rifampin, thiamphenicol, tinidazole, dapsone and clofazimine.Antibiotic example is listed in table 15.

Antiviral agent includes but not limited to Abacavir, acyclovir, acycloguanosine, adefovir ester, amantadine, amprenavir, Ampligen (Ampligen), Arbidol, Reyataz R, Atripla, Bo Xipuwei (Boceprevir), cidofovir, Combivir, DRV, Delavirdine, didanosine, V-1326, edoxudine, efavirenz, emtricitabine, En Fuwei, Entecavir, Famciclovir, Fomivirsen, fosamprenavir, FOSCARNET, phosphine ethanol, ganciclovir, ibacitabine, isoprinosine, iodoxuridine, Imiquimod, Indinavir, inosine, type iii interferon, interferon type Ⅱ, I type Interferon, rabbit, lamivudine, rltonavir, loviride, MVC, Moroxydine, viracept see nelfinaivr, nevirapine, Nexavir, Oseltamivir, polyoxyethylene glycol interferon α-2 A, Penciclovir, Peramivir, pleconaril, podophyllotoxin, Merck, ribavirin, Rimantadine, ritonavir, Pyramidine, Saquinavir, stavudine, tea tree oil, tynofovir, tenofovir disoproxil, tipranavir, floxuridine, Trizivir, tromantadine, Troyes reaches, valacyclovir, valganciclovir, Vicriviroc, vidarabine, Viramidine, zalcitabine, zanamivir and zidovudine.

Table 15: the example of antibiotic medicine and structured sort thereof

In one embodiment, the experimenter suffers from the HIV infection.Can assess one or more biomarkers from the vesica from described experimenter, such as, but not limited to, p24 antigen, TNF-α, TNFR-II, CD3, CD14, CD25, CD27, Fas, FasL, β2-microglobulin, mopterin, HIV RNA and HLA-B*5701.According to one or more features of described one or more biomarkers, described experimenter can be defined as to respondent or non-respondent for treatment (such as, but not limited to zidovudine, didanosine, zalcitabine, stavudine, lamivudine, Saquinavir, ritonavir, Indinavir, Nevirane, viracept see nelfinaivr, U-90152, stavudine, efavirenz, etravirine, enfuirtide, darunavir, Abacavir, An Ruinawei, Lonavir/ ritonavir, tynofovir, tipranavir or their combination).

Therefore, can be imprinted as the experimenter who suffers from infectious diseases or situation according to the biology of experimenter's vesica and select treatment.

Neuroscience

The assessment of vesica also be can be used for to the treatment diagnosis of sacred disease, such as, multiple sclerosis (MS) for example, Parkinson's disease (PD), alzheimer's disease (AD) (non-inflammatory or inflammatory), schizophrenia, bipolar disorder, dysthymia disorders, autism, prion disease, Pick's disease, dull-witted, Huntington Chorea (HD), mongolism, cerebrovascular disease, the Rasmussen encephalitis, viral meningitis, neuropsychopathy systemic lupus erythematous (NPSLE), amyotrophic lateral sclerosis, creutzfeldt-Jacob disease, the Gerstmann-Straussler-Scheinker disease, Transmissible spongiform encephalopathy, ischemical reperfusion injury (for example apoplexy), cerebral trauma, infected by microbes or chronic fatigue syndrome.

Nervous disorder includes but not limited to inflammatory diseases, the heredity degenerative disease of central nervous system, pain, other have a headache syndrome, other imbalance of central nervous system and imbalance of peripheral nervous system of central nervous system.The inflammatory diseases of central nervous system includes but not limited to, bacterial meningitis, meningitis, Haemophilus meningitis, the bacterium meningitis, due to the meningitis due to other organism, crypotococcal, the unknown cause meningitis, encephalitis, myelitis and encephalomyelitis, postinfectious encephalitis, not clear encephalitis, encephalic and intraspinal abscess, the phlebitis of dlural sinus and thrombophlebitis, Cerebral venous sinus thrombosis forms, the anaphase effect of intracranial abscess or pyogenic infection, somnopathy, not clear organic insomnia, due to the insomnia due to the medical condition of in addition classification and due to the insomnia due to mental disorder.The brain Childhood that the heredity of central nervous system and degenerative disease including but not limited to usually show is degenerated, leukodystrophy, galactosylceramide beta-galactosidase deficiency (krabbe disease), Pelizaeus Merzbacher disease, the cephalopin deposition, Tay Sachs disease, other brain is degenerated, alzheimer's disease, Pick's disease, senile brain is degenerated, communicating hydrocephalus, obstructive hydrocephalus, idiopathic normal pressure hydrocephalus, other brain is degenerated, Reye syndrome, dementia with Lewy body disease, so-called mild cognition impairment, Parkinson's disease, former Parkinson's disease, other extrapymidal disease and abnormal motion imbalance, other degenerative disorders of basal ganglion, OPCA, shy-Drager syndrome (shy drager syndrome), essential tremor/familial tremor, myoclonus, myoclonic epilepsy (lafora ' s disease), unverricht disease (unverricht disease), huntington's chorea, torsion dysmyotonia, blepharospasm, other reaches not clear extrapymidal disease and abnormal motion imbalance, other extrapymidal disease and abnormal motion imbalance, many moving legs, serotonin syndrome, spinocerebellar disease, family ataxia, spinocebellar ataxia, hereditary spastic paraplegia, the primary cerebellum degeneration, other cerebellar ataxia, the cerebellar ataxia disease of classifying in addition, other spinocerebellar disease, ataxia telangiectasia, the cortex spinal cord is degenerated, not clear spinocerebellar disease, the ventricornu cell disease, motor neurone disease, amyotrophic lateral sclerosis, progressive myatrophy, progressive bulbar palsy, pseudobulbar paralysis, primary lateral sclerosis, other motor neurone disease, other diseases of spinal cord, syringomyelia and syringobulbia, autonomic other imbalance, idiopathic periphery autonomic neuropathy, not clear idiopathic periphery autonomic neuropathy, carotid sinus syndrome, other idiopathic periphery autonomic neuropathy, the periphery autonomic neuropathy of the imbalance of classifying in addition, sympathetic reflex dystrophy, autonomic dysreflexia and autonomic not clear imbalance.

Pain includes but not limited to, acute and chronic pain and the chronic pain syndrome of central pain syndrome, acute pain, chronic pain, Tumor-assaciated.Other headache syndrome includes but not limited to, cluster headache and the headache of other trident autonomic nerve, not clear cluster headache syndrome, ictal cluster headache, chronic cluster headache, ictal paroxysmal hemicrania, chronic paroxysmal hemicrania, short lasting one-sided class neurodynia headache (sign an undertaking film congested and shed tears), other trident autonomic nerve headache, tension headache, not clear tension headache, Therapy on Episode Tension-type Headache, chronic tension-type headache, post-traumatic headache, not clear post-traumatic headache, after acute injury, have a headache, after chronic trauma, have a headache, the not drug-induced headache of classification in addition, concurrency headache syndrome, the continuity migraine, new persistence headache every day, the primary thunderclap headache, other concurrency headache syndrome, the headache syndrome that other is clear and definite, the sleep headache, the relevant headache of sexual behaviour, primary cough headache, primary does all one can to have a headache and primary shouting pain headache.

Other imbalance of central nervous system includes but not limited to, multiple sclerosis, other demyelinating disease of central nervous system, optic neuromyelitis, schilder's disease (schilder ' s disease), the horizontal myelitis of acute myelitis, hemiplegia, flaccid hemiplegia, spastic hemiplegia, cerebral palsy of children, the congenital paraplegia cerebral paralysis, the congenital hemiplegia cerebral paralysis, the tetraplegia cerebral paralysis, other paralytic syndrome, tetraplegia and quadriparesis, paraplegia, the upper limbs diplegia, lower limb monoplegia, upper limbs monoplegia, not clear monoplegia, cauda equina syndrome, the paralytic syndrome of the state locking that other is clear and definite, epilepsy, intractable epilepsy, the tetanic clonic epilepsy of stateless, the stateless temporal epilepsy, stateless is failed to understand epilepsy, migraine, classical non-refractory migraine, common non-refractory migraine, non-intractable cluster headache, not clear non-refractory migraine, damping off and narcolepsy, without dampinging off narcolepsy, cerebral cyst, hypoxic brain injury, pseudotumor cerebri, not clear encephalopathic, metabolic encephalopathy, the compression of brain, cerebral edema, rear thecal puncture, rear endocranium puncture headache, cerebrospinal rhinorrhea and toxic encephalopthy.

The imbalance of peripheral nervous system includes but not limited to, the trigeminal nerve imbalance, trigeminal neuralgia, facial nerve disorders, bell's palsy, other cranial nerve imbalance, nerve root and neuroplexus imbalance, syndrome of chest outlet, phantom limb, mononeuritis of upper limb and canalis carpi mononeuritis multiplex, mononeuritis of lower limb, the sciatic nerve pathology, meralgia paraesthetica, other femoral nerve pathology, Ce popliteal DPN, Nei Ce popliteal DPN, tarsal tunnel syndrome, the nervus plantaris pathology, the Mo Dunshi neuroma, lower limb are failed to understand mononeuritis, not clear position mononeuritis, heredity and idiopathic peripheral neuropathy, inflammation and toxic neuropathy, Guillain Barre syndrome, polyneuropathy, the alcohol polyneuropathy, the muscular nerve imbalance, follow the myasthenia gravis of deterioration, without worsening myasthenia gravis, muscular dystrophy and other myopathy, optimum congenital myopathy, central core disease, the central nucleus myopathy, the myotube myopathy, nemaline body disease (nemaline body disease) and hereditary amyotrophy.

Can assess the biological marking of vesica and think that the experimenter provides the treatment diagnosis.The biological marking of described vesica can comprise one or more biomarkers, such as, but not limited to, disclosed those biomarkers of following table:

neuroscience: the biological marking

Can assess the biological marking of vesica and think that the experimenter provides the treatment diagnosis.The biological marking of described vesica can comprise one or more biomarkers, such as, but not limited to, listed biomarker in Fig. 1,45,46,47,48 and 49.The biological marking of described vesica can comprise one or more biomarkers, it includes but not limited to amyloid beta, ICAM-1 (rodents), CGRP (rodents), TIMP-1 (rodents), CLR-1 (rodents), HSP-27 (rodents), FABP (rodents), ATP5B, ATP5H, ATP6V1B, DNM1, NDUFV2, NSF, PDHB, FGF2, ALDH7A1, AGXT2L1, AQP4, PCNT2, FGFR1, FGFR2, FGFR3, AQP4, the sudden change of Dysbindin, DAOA/G30, DISC1, neuregulin-1, IFITM3, SERPINA3, GLS, ALDH7A1, BASP1, OX42, ED9, Apolipoprotein D (rodents), miR-7, miR-24, miR-26b, miR-29b, miR-30b, miR-30e, miR-92, miR-195, miR-181b, DISC1, dysbindin, neuregulin-1, thrombotonin (seratonin) 2a acceptor and NURR1.

neuroscience: nursing standard

The biological marking of vesica of the sample of the experimenter from suffering from nervosa imbalance or disease, vesica amount or both are measured and can be used for selecting nursing standard for described experimenter.Nervosa imbalance or disease can be treated according to the symptom relevant to described situation.Nursing standard can comprise, for example, and medicine.Medicine can include but not limited to, acetylsalicylic acid, Dipyridamole, naratriptan, apomorphine, E2020, the oxysuccinic acid almotriptan, Rufinamide, Bromfenac, carbatrol, cenestin, Tadalafei, clonazepam, Entacapone, Glatiramer acetate, pemoline, two valproic acids, difluprednate, Zolpidem Tartrate, the tartrate profit is cut down the bright of this, dexmethylphenidate, the succsinic acid SB 209509, zinc acetate, sumatriptan, paliperidone, iontocaine, morphine, Levetiracetam, lamotrigine, Vardenafil, lignocaine, Lunesta, the phosphorus propofol disodium, lyrica, rizatriptan benzoate, meropenem, Ritalin, dihydroetgotamine, pramipexole, rimabotulinumtoxin B, naltrexone, Memantine hydrochloride, for the dagger-axe spit of fland, gabapentin), hydrocodone, mitoxantrone, l-modafinil, oxycodone, pramipexole, samariumlexidronam 153, interferon beta-1a, dexfenfluramine, Hydrogen bromide Yi Liputan, galanthamine hydrobromide, ropinirole hydrochloride, Riluzole, Ramelteon, selegiline, valproic acid, tomoxetine, tolcapone, Carbamzepine, topiramate, oxcarbazepine, natalizumab, paracetamol, U-26225A, midazolam, draw section's amine, Visipaque 320, two methylsulfonic acids rely Dextrofenfluramine, tetrabenazine, sodium hydroxybutyrate, tizanidine hydrochloride, zolmitriptan and zonisamide.

Other treatment that can select according to experimenter's vesica spectrum comprises the treatment of listing for the experimenter who suffers from multiple sclerosis in table 14; The treatment of listing for suffering from parkinsonian experimenter in table 16; The treatment of perhaps listing for the experimenter who suffers from dysthymia disorders in table 17.

Table 16: for the drug categories of Parkinson's disease treatment

Table 17: for the drug categories for the treatment of depression

In one embodiment, can select for the experimenter who suffers from alzheimer's disease treatment.Can assess one or more biomarkers from the vesica from described experimenter, such as, but not limited to amyloid-beta, amyloid precursor protein (APP), APP670/671, APP693, APP692, APP715, APP716, APP717, APP723, early ageing albumen 1, early ageing albumen 2, cerebrospinal fluid amyloid beta protein 42 (CSF-A β 42), cerebrospinal fluid amyloid beta protein 40 (CSF-A β 40), F2 isoprostane, 4-hydroxyl nonenal, F4neuroprostane and propenal.According to one or more features of described one or more biomarkers, described experimenter can be defined as to respondent or non-respondent for treatment (cutting down this bright, tacrine or its combination such as, but not limited to E2020, lycoremine, memantine, profit).

In another embodiment, can select treatment for suffering from parkinsonian experimenter.Can assess one or more biomarkers from the vesica from described experimenter, such as, but not limited to α synapse nucleoprotein, PARK7 (DJ-1), S phase kinase-associated protein 1A (p19A/SKP1A), heat shock protein 70 kDa, AMP regulation and control phosphoric acid albumen (ARPP-21), vesica monoamine member 2 (VMAT2), alcoholdehydrogenase 5 (ADH5), aldehyde dehydrogenase 1A1 (ALDH1A1), Ai Geer 9 homologues 1 (EGLN1), Protocollagen prolyl hydroxylase 2 (PHD2) and hypoxia inducible factor (HIF).According to one or more features of described one or more biomarkers, described experimenter can be defined as to respondent or non-respondent for treatment (such as, but not limited to table 16 listed those).

In another embodiment, can select treatment for suffering from parkinsonian experimenter.Can assess one or more biomarkers from the vesica from described experimenter, such as, but not limited to CRP, TNF, IL-6, S100B and MMP.According to one or more features of described one or more biomarkers, described experimenter can be defined as to respondent or non-respondent for treatment.

Therefore, can be imprinted as according to the biology of experimenter's vesica and suffer from neural conditions associated or experimenter neural status or disease and select treatment.

The biological marking is found

System and method provided herein can be used for identifying the new bio marking of vesica, such as for phenotype is diagnosed, one or more new bio marks of prognosis or treatment diagnosis.The biological marking that can separate from the experimenter with phenotype in one embodiment, one or more vesicas and definite described one or more vesicas.The described biological marking can compare with the experimenter of the described phenotype of tool not.Can measure two kinds of differences between the biological marking and use it for and form the new bio marking.The described new bio marking can be subsequently has described phenotype or the described phenotype of tool not for another experimenter is accredited as.

From the biological marking of the experimenter with particular phenotype can with carry out diversity ratio from the experimenter's of the described particular phenotype of tool the biological marking not.Described one or more differences can be the difference of the arbitrary characteristics of described vesica.For example, between the biological marking and the biological marking from the experimenter who does not have described particular phenotype of the experimenter from having particular phenotype, in the transformation period of the level of the vesica in sample and amount, vesica, the circulation of vesica, active or its arbitrary combination of the metabolic half life of transformation period, vesica or vesica can be different.

In some embodiments, between the biological marking and the biological marking from the experimenter who does not there is described particular phenotype of the experimenter from thering is particular phenotype, one or more biomarker differences.For example, between the biological marking and the biological marking from the experimenter who does not there is described particular phenotype of the experimenter from thering is particular phenotype, the expression level of one or more biomarkers, existence, do not exist, associated or its arbitrary combination of sudden change, varient, number of copies variation, brachymemma, repetition, modification, molecule can be different.Described biomarker can be any biomarker disclosed herein, or can be used for the biomarker of characterising biological entity, it comprises circulating biological mark (such as protein or microRNA), vesica or is present in the component in vesica or on vesica, such as any nucleic acid (as RNA or DNA), protein, peptide, polypeptide, antigen, lipid, carbohydrate or proteoglycan.

In one aspect, the invention provides the method for finding the new bio marking, comprise the biomarker between two or more sample sets is compared to identify the biomarker that demonstrates difference between sample sets.Can be with group's form (panel format) assessment multiple markers to improve potentially the performance of single mark.In some embodiments, assess described multiple markers in multiple mode.Can use statistic discriminance analysis as herein described and classifying method to assess the ability of single mark and each group of mark group differentiation.The optimum group of mark can be used as for characterizing the biological marking of the phenotype of analyzing, such as the diagnosis, prognosis or the treatment diagnosis that provide disease or situation.Optimization can be carried out based on multiple standards, includes but not limited to maximize sensitivity under ROC AUC, accuracy, specific specificity or the specificity under particular sensitivity.Described group can comprise from polytype biomarker.For example, the described biological marking can comprise the vesica antigen that can be used for target acquisition vesica colony, and the described biological marking can further comprise the useful load mark in described vesica colony, and it includes but not limited to microRNA, mRNA or soluble protein.Optimum combination can be confirmed as the combination that has vesica antigen and the useful load mark of the highest ROC AUC value when comparing two kinds of situations.As another example, the circulating biological mark (such as circulating protein matter and/or microRNA) that can not be obtained by the separation exosome by assessment vesica colony and assessment is determined the described biological marking.

Described phenotype can be the listed any phenotype of this paper, as, list in above in " phenotype " part.For example, described phenotype can be proliferative imbalance, and such as cancer or non-malignant growth, perinatal period or gestation are conditions associated, infectious diseases, nervous disorder, cardiovascular disorder, inflammatory diseases, Immunological diseases or autoimmune disease.Described cancer includes but not limited to lung cancer, non-small cell carcinoma, small cell lung cancer (comprises small cell carcinoma (oat-cell carcinoma), minicell/maxicell mixed carcinoma and plyability small cell carcinoma), colorectal carcinoma, mammary cancer, prostate cancer, liver cancer, carcinoma of the pancreas, the cancer of the brain, kidney, ovarian cancer, cancer of the stomach, melanoma, osteocarcinoma, the cancer of stomach, mammary cancer, glioma, glioblastoma multiforme, hepatocellular carcinoma, the corpora mammillaria kidney, squamous cell carcinoma of the head and neck, leukemia, lymphoma, myelomatosis or other noumenal tumour.

Can assess any biomarker type as herein described or particular organisms mark to find the new bio marking.In one embodiment, select the biomarker for finding to comprise the listed cell-specific biomarker of this paper, it includes but not limited to listed gene and microRNA in Fig. 1-60, table 9-11 or table 27-41.Described biomarker can comprise the relevant target of one or more medicines, such as ABCC1, ABCG2, ACE2, ADA, ADH1C, ADH4, AGT, AR, AREG, ASNS, BCL2, BCRP, BDCA1, β III tubulin, BIRC5, B-RAF, BRCA1, BRCA2, CA2, caveolin, CD20, CD25, CD33, CD52, CDA, CDKN2A, CDKN1A, CDKN1B, CDK2, CDW52, CES2, CK 14, CK 17, CK 5/6, c-KIT, c-Met, c-Myc, COX-2, cyclin D1, DCK, DHFR, DNMT1, DNMT3A, DNMT3B, CAM 120/80, ECGF1, EGFR, the EML4-ALK syzygy, EPHA2, epiregulin, ER, ERBR2, ERCC1, ERCC3, EREG, ESR1, FLT1, folacin receptor, FOLR1, FOLR2, FSHB, FSHPRH1, FSHR, FYN, GART, GNRH1, GNRHR1, GSTP1, HCK, HDAC1, hENT-1, Her2/Neu, HGF, HIF1A, HIG1, HSP90, HSP90AA1, HSPCA, IGF-1R, IGFRBP, IGFRBP3, IGFRBP4, IGFRBP5, IL13RA1, IL2RA, KDR, Ki67, KIT, K-RAS, LCK, LTB, lymphotoxin-beta-receptor, LYN, MET, MGMT, MLH1, MMR, MRP1, MS4A1, MSH2, MSH5, Myc, NFKB1, NFKB2, NFKBIA, ODC1, OGFR, p16, p21, p27, p53, p95, PARP-1, PDGFC, PDGFR, PDGFRA, PDGFRB, PGP, PGR, PI3K, POLA, POLA1, PPARG, PPARGC1, PR, PTEN, PTGS2, RAF1, RARA, RRM1, RRM2, RRM2B, RXRB, RXRG, SIK2, SPARC, SRC, SSTR1, SSTR2, SSTR3, SSTR4, SSTR5, Survivin, TK1, TLE3, TNF, TOP1, TOP2A, TOP2B, TS, TXN, TXNRD1, TYMS, VDR, VEGF, VEGFA, VEGFC, VHL, YES1 and ZAP70.Described biomarker can comprise one or more general vesica marks, one or more cell-specific vesica marks and/or one or more disease specific vesica marks.

The biomarker of finding for the biological marking can comprise the mark relevant to vesica usually, it includes but not limited to HSPA8, CD63, Actb, GAPDH, CD9, CD81, ANXA2, HSP90AA1, ENO1, YWHAZ, PDCD6IP, CFL1, SDCBP, PKN2, MSN, MFGE8, EZR, YWHAG, PGK1, EEF1A1, PPIA, GLC1F, GK, ANXA6, ANXA1, ALDOA, ACTG1, TPI1, LAMP2, HSP90AB1, DPP4, YWHAB, TSG101, PFN1, LDHB, HSPA1B, HSPA1A, GSTP1, GNAI2, GDI2, CLTC, ANXA5, YWHAQ, TUBA1A, THBS1, PRDX1, LDHA, LAMP1, one or more in CLU and CD86.Described biomarker can further comprise CD63, GAPDH, CD9, CD81, ANXA2, ENO1, SDCBP, MSN, MFGE8, EZR, GK, ANXA1, LAMP2, DPP4, TSG101, HSPA1A, GDI2, CLTC, LAMP1, Cd86, ANPEP, TFRC, SLC3A2, RDX, RAP1B, RAB5C, RAB5B, MYH9, ICAM1, FN1, RAB11B, PIGR, LGALS3, ITGB1, EHD1, CLIC1, ATP1A1, ARF1, RAP1A, P4HB, MUC1, KRT10, HLA-A, FLOT1, CD59, C1orf58, BASP1, one or more in TACSTD1 and STOM.Other biomarker can be selected from disclosed biomarker in the ExoCarta database, and described database is found in exocarta.ludwig.edu.au, and it discloses protein and the RNA molecule of identifying in exosome.Also referring to Mathivanan and Simpson, ExoCarta:A compendium of exosomal proteins and RNA.Proteomics.2009 (21): 4997-5000 on November 9.

The biomarker of finding for the biological marking can comprise the mark relevant to vesica usually, and it includes but not limited to A33, a33 n15, AFP, ALA, ALIX, ALP, annexin V, APC, ASCA, ASPH (246-260), ASPH (666-680), ASPH (A-10), ASPH (D01P), ASPH (D03), ASPH (G-20), ASPH (H-300), AURKA, AURKB, B7H3, B7H4, BCA-225, BCNP1, BDNF, BRCA, CA125 (MUC16), CA-19-9, C-Bir, CD1.1, CD10, CD174 (Lewis y), CD24, CD44, CD46, CD59 (MEM-43), CD63, CD66e CEA, CD73, CD81, CD9, CDA, CDAC1 1a2, CEA, C-Erb2, C-erbB2, CRMP-2, CRP, CXCL12, CYFRA21-1, DLL4, DR3, EGFR, Epcam, EphA2, EphA2 (H-77), ER, ErbB4, EZH2, FASL, FRT, FRT c.f23, GDF15, GPCR, GPR30, Gro-α, HAP, HBD 1, HBD2, HER 3 (ErbB3), HSP, HSP70, hVEGFR2, iC3b, IL 6 Unc, IL-1B, IL6 Unc, IL6R, IL8, IL-8, INSIG-2, KLK2, L1CAM, LAMN, LDH, MACC-1, MAPK4, MART-1, MCP-1, M-CSF, MFG-E8, MIC1, MIF, MIS RII, MMG, MMP26, MMP7, MMP9, MS4A1, MUC1, MUC1seq1, MUC1 seq11A, MUC17, MUC2, Ncam, NGAL, NPGP/NPFF2, OPG, OPN, p53, p53, PA2G4, PBP, PCSA, PDGFRB, PGP9.5, PIM1, PR (B), PRL, PSA, PSMA, PSME3, PTEN, R5-CD9Tube 1, Reg IV, RUNX2, SCRN1, seprase, SERPINB3, SPARC, SPB, SPDEF, SRVN, STAT 3, STEAP1, TF (FL-295), TFF3, TGM2, TIMP-1, TIMP1, TIMP2, TMEM211, TMPRSS2, TNF-α, Trail-R2, Trail-R4, TrKB, TROP2, Tsg 101, TWEAK, UNC93A, one or more in VEGF A and YPSMA-1.Described biomarker can comprise NSE, TRIM29, CD63, CD151, ASPH, LAMP2, TSPAN1, SNAIL, CD45, CKS1, NSE, FSHR, OPN, FTH1, PGP9, annexin 1, SPD, CD81, EPCAM, PTH1R, CEA, CYTO 7, CCL2, SPA, KRAS, TWIST1, AURKB, MMP9, P27, MMP1, HLA, HIF, CEACAM, CENPH, BTUB, INTG b4, EGFR, NACC1, CYTO 18, NAP2, CYTO 19, ANNEXIN V, TGM2, ERB2, BRCA1, B7H3, SFTPC, PNT, NCAM, MS4A1, P53, INGA3, MUC2, SPA, OPN, CD63, CD9, MUC1, UNCR3, PAN ADH, HCG, TIMP, PSMA, GPCR, RACK1, PCSA, VEGF, BMP2, CD81, CRP, PRO GRP, B7H3, MUC1, M2PK, CD9, one or more in PCSA and PSMA.Described biomarker can comprise one or more in TFF3, MS4A1, EphA2, GAL3, EGFR, N-gal, PCSA, CD63, MUC1, TGM2, CD81, DR3, MACC-1, TrKB, CD24, TIMP-1, A33, CD66CEA, PRL, MMP9, MMP7, TMEM211, SCRN1, TROP2, TWEAK, CDACC1, UNC93A, APC, C-Erb, CD10, BDNF, FRT, GPR30, P53, SPR, OPN, MUC2, GRO-1, tsg 101 and GDF15.In embodiments, be included in one or more biomarkers shown in Figure 99,100,108A-C, 114A and/or 115A-E for the biomarker of finding the biological marking.

The technician should be appreciated that this paper openly or is used in any mark compared between two target sample or sample sets and can be used for finding the new bio marking for any given biology situation that can be compared.

Described one or more differences can be used to form the new bio marking of described particular phenotype subsequently, such as the diagnosis to situation, to the diagnosis in stage of disease or situation, to the prognosis of situation or to the treatment diagnosis of situation.The described new bio marking can be subsequently for identifying described phenotype other experimenter.Can determine for the biological marking of new experimenter's vesica and itself and the described novel marking are compared to determine whether described experimenter has the particular phenotype that the described new bio marking is therefrom identified.

For example, the biological marking of suffering from the experimenter of cancer can compare with not cancered another experimenter.Any difference can be used for being formed for the new bio marking of the diagnosis of described cancer.In another embodiment, the biological marking of suffering from the experimenter of terminal cancer can compare with another experimenter who suffers from than early-stage cancer.Any difference can be used for forming the new bio marking with the classification for to carcinoma stage.In another embodiment again, the biological marking of suffering from the experimenter of terminal cancer can compare with the experimenter who suffers from than early-stage cancer.Any difference can be used for forming the new bio marking with the classification for to carcinoma stage.

In one embodiment, described phenotype is drug resistance or non-reacted for therapy.The biological marking that can from the non-respondent to particular treatment, separate one or more vesicas and definite described vesica.Available from the biological marking of described non-respondent's vesica, can compare with the biological marking of vesica from the respondent.Can be relatively from described non-respondent's the biological marking and from the difference between described respondent's the biological marking.Described one or more differences can be the difference of the arbitrary characteristics of vesica.For example, between the biological marking from non-respondent and the biological marking from the respondent, active or its arbitrary combination of the metabolic half life of the transformation period of the level of the vesica of described sample and amount, vesica, the circulating half-life of vesica, vesica, vesica can be different.

In some embodiments, between the biological marking from non-respondent and the biological marking from the respondent, one or more biomarker differences.For example, between the biological marking from non-respondent and the biological marking from the respondent, the expression level of one or more biomarkers, existence, do not exist, associated or its arbitrary combination of sudden change, varient, number of copies variation, brachymemma, repetition, modification, molecule can be different.

In some embodiments, described difference can be the medicine that exists in vesica or the amount of drug metabolite.Can use therapeutical agent treatment respondent and non-respondent.Can carry out the comparison between described respondent's the biological marking and described non-respondent's the biological marking, the medicine existed in the vesica from described respondent or the amount of drug metabolite can be different from the medicine that exists in the vesica from described non-respondent or the amount of drug metabolite.Described difference also can be the transformation period of described medicine or drug metabolite.The amount of described medicine or drug metabolite or the difference of transformation period can be used for forming the new bio marking with for the identification of non-respondent and respondent.

The vesica that can be used for method and composition as herein described can be found by utilizing its plysiochemical feature.For example, vesica can according to its size find, for example, by the 30-120nm diameter range inner filtration biological substance known.Discover method based on big or small can be separated for vesica, such as differential centrifugation, sucrose gradient centrifugation or filtration.

Vesica can be found according to its minute subconstiuent.Discover method based on molecular characterization includes but not limited to, uses the immunity that the antibody of the molecule that identification is relevant to vesica carries out to separate.For example, the surface molecular relevant to vesica includes but not limited to, MHC-II molecule, CD63, CD81, LAMP-1, Rab7 or Rab5.

Multiple technologies as known in the art can be applicable to checking and the evaluation of vesica.Can be used for the checking of vesica and the technology of evaluation and include but not limited to, Western trace, electron microscopy, immunohistochemistry, Immunoelectron microscopy, FACS (fluorescence activated cell sorting), electrophoresis (1 dimension, 2 dimensions), liquid chromatography, mass spectrum, MALDI-TOF (substance assistant laser desorpted/ionization flight time), ELISA, LC-MS-MS and nESI (nanometer electron spray(ES)).For example United States Patent (USP) 2009/0148460 has been described the purposes of ELISA method for the identification of vesica.United States Patent (USP) 2009/0258379 has been described the separation of membrane vesicle from biological liquid.

Can further analyze vesica for one or more nucleic acid, lipid or polypeptide (such as surface protein or peptide, or protein or peptide in vesica).Can identify candidate's peptide by various technology (comprising the mass spectrum with purification process coupling as liquid chromatography).Isolated peptides is also identified its sequence by order-checking subsequently.Computer program based on peptide accurate mass forecasting sequence also can be used for disclosing the sequence of separating from the peptide of vesica.For example, the LTQ-Orbitrap mass spectrum can be used for the peptide sequencing of highly sensitive and high accuracy.The LTQ-Orbitrap method is described (Simpson etc., Expert Rev.Proteomics 6:267-283,2009), and it is incorporated to this paper in full by reference.

Vesicle

In addition, this paper also provides the vesica of the separation with particular organisms marking.The vesica separated can comprise to one or more biomarkers or the biological marking specific cell type-specific or for characterizing phenotype, as described above.For example, the vesica of separation can comprise one or more biomarkers, for example CD63, EpCam, CD81, CD9, PCSA, PSMA, B7H3, TNFR, MFG-E8, Rab, STEAP, 5T4 or CD59.The vesica separated can comprise one or more following biomarker: EpCam, CD9, PCSA, CD63, CD81, PSMA, B7H3, PSCA, ICAM, STEAP and EGFR.In one embodiment, described vesica is EpCam+, CK+, CD45-.The vesica of described separation in its surface or can have these one or more biomarkers in described vesica.In addition, the vesica of described separation can also comprise one or more miRNA, for example miR-9, miR-629, miR-141, miR-671-3p, miR-491, miR-182, miR-125a-3p, miR-324-5p, miR-148B or miR-222.In one embodiment, described vesica comprises one or more miRNA, for example miR-548c-5p, miR-362-3p, miR-422a, miR-597, miR-429, miR-200a and miR-200b.In another embodiment again, described vesica comprises one or more miRNA, for example miR-92a-2 ^*, miR-147, miR-574-5p.The vesica separated can comprise biomarker, CD66 for example, and further comprise one or more biomarkers that are selected from EpCam, CD63 or CD9.The vesica separated can also comprise fusion gene or protein, for example TMRSSG2:ERG.

The vesica separated can also comprise one or more biomarkers, wherein be derived from normal cell (, cell from the experimenter who does not have the target phenotype) separation vesica is compared, and the expression level of described one or more biomarkers of the vesica of separation is higher, lower or identical.For example, the vesica separated can comprise and is selected from: one or more biomarkers in B7H3, PSCA, MFG-E8, Rab, STEAP, PSMA, PCSA, 5T4, miR-9, miR-629, miR-141, miR-671-3p, miR-491, miR-182, miR-125a-3p, miR-324-5p, miR-148b and miR-222, wherein with being derived from Normocellular vesica, compare, the expression level of one or more biomarkers of the vesica of described separation is higher.The vesica of described separation can comprise and is selected from least 2,3,4,5,6,7,8,9,10,11,13,14,15,16,17,18 in described group or 19 kind of biomarker.The vesica of described separation can further comprise one or more biomarkers that are selected from EpCam, CD63, CD59, CD81 or CD9.

The vesica separated can comprise biomarker PCSA, EpCam, CD63 and CD8; Biomarker PCSA, EpCam, B7H3 and PSMA.The vesica separated can comprise biomarker miR-9, miR-629, miR-141, miR-671-3p, miR-491, miR-182, miR-125a-3p, miR-324-5p, miR-148b and miR-222.

This paper also provides the composition of the vesica that comprises separation.Described composition can comprise the vesica of one or more separation.For example, described composition can comprise one or more colonies of multiple vesica or vesica.

Described composition can be for the obvious enrichment of vesica.For example, described composition does not exist cell debris, cell, non-exosome protein, peptide or nucleic acid (biomolecules for example do not comprised in described vesica) basically.Described cell debris, cell, non-exosome protein, peptide or nucleic acid may be present in biological sample together with vesica.Basically do not exist the composition of cell debris, cell, non-exosome protein, peptide or nucleic acid (for example non-biological molecule be contained in described vesica) to obtain by any method disclosed herein, for example, by using one or more wedding agents or the trapping agent for one or more vesicas.Described vesica can account at least 30,40,50,60,70,80,90,95 or 99% of the weight of total composition or quality.The vesica of described composition can be vesica heterogeneous or homogeneous colony.For example, the vesica colony of homogeneous comprises for one or more character or feature the vesica for homogeneous.For example, described one or more features can be selected from: one or more identical biomarkers, the similar or consistent biological marking, the vesica that is derived from identical cell type, specific size and their combination basically.

Therefore, in some embodiments, the vesica colony that described composition comprises obvious enrichment.Described composition can be enrichment for the vesica colony of at least 30,40,50,60,70,80,90,95 or 99% homogeneous with regard to one or more character or feature.For example, described one or more features can be selected from: one or more identical biomarkers, the similar or consistent biological marking, the vesica that is derived from identical cell type, specific size and their combination basically.For example, described vesica colony can be by all thering is the specific biological marking, there is identical biomarker, there is identical biomarker combinations or be derived from identical cell type and become homogeneous.In some embodiments, described composition comprises the vesica colony of homogeneous basically, for example, has the particular organisms marking, is derived from specific cell or the colony of these two.

Vesica colony can comprise one or more identical biomarkers.Described biomarker can be any composition, for example, and any nucleic acid (for example RNA or DNA), protein, peptide, polypeptide, antigen, lipid, carbohydrate or proteoglycan.For example, each vesica in colony can comprise one or more identical or consistent biomarkers.In some embodiments, each vesica comprises identical 1,2,3,4,5,6,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,25,50,75 or 100 kind of biomarker.Described one or more biomarkers can be selected from Fig. 1,3-60.

The vesica colony that comprises identical or consistent biomarker can refer to each vesica in described colony there is the existence of identical described biomarker or do not exist, expression level, mutation status or modification.For example, the vesica colony of enrichment can comprise identical biomarker that each vesica wherein has existence, shortage identical biomarker, identical biomarker expression level, identical biomarker is modified or the vesica of identical biomarker sudden change.The identical expression level of biomarker can specified amount or qualitative measure, for example, with reference level, compare, the vesica in described colony for biomarker be express not enough, cross express or there is identical expression level.

Perhaps, the identical expression level of biomarker can be the numerical value of representative similar biomarker expression for each vesica in colony.For example, the level of the copy number of the miRNA of each vesica, the amount of protein or mRNA can quantitatively be similar to for each vesica in colony, make the amount of each other vesica in the quantitative value of each vesica and described colony differ ± 1,2,3,4,5,6,7,8,9,10,15 or 20%, as long as this variation is suitable.

In some embodiments, the vesica colony that described composition comprises obvious enrichment, wherein the vesica in described enrichment colony has the basically similar or consistent biological marking.The described biological marking can comprise one or more vesica features, for example Time evaluation, circulating vesica transformation period, the metabolic half life of vesica or the activity of vesica of the level of vesica or amount, the variation of vesica transformation period.The described biological marking can also comprise the existence of biomarker or do not exist, expression level, mutation status or modification, all as described herein those.

In described colony, the biological marking of each vesica is can at least 30,40,50,60,70,80,90,95 or 99% identical.In some embodiments, the biology of each vesica is imprinted as 100% identical.In the colony of described enrichment, the biological marking of each vesica can have identical a kind, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds, 9 kinds, 10 kinds, 11 kinds, 12 kinds, 13 kinds, 14 kinds, 15 kinds, 16 kinds, 17 kinds, 18 kinds, 19 kinds, 20 kinds, 25 kinds, 50 kinds, 75 kinds or 100 kinds of features.For example, in the colony of enrichment, the biological marking of vesica can exist and the 3rd biological marker expression deficiency for the first biomarker existence, the second biomarker.Another vesica in identical colony can, for 100% identical, have identical the first and second biomarkers and the 3rd biological marker expression deficiency of existence.Perhaps, the vesica in same community can have the first and second identical biomarkers, but there is no the expression deficiency of the 3rd biological mark.

In some embodiments, the vesica colony that described composition comprises obvious enrichment, wherein said vesica is derived from identical cell type.For example, described vesica can all be derived from the cell of particular organization, from the cell of cell, circulating tumor cell or the parent of specific objective tumour or target disease tissue or fetus origin.Described vesica can all be derived from tumour cell.Described vesica can all be derived from lung, pancreas, stomach, intestines, bladder, kidney, ovary, testis, skin, colorectum, mammary gland, prostate gland, brain, oesophagus, liver, placenta or fetal cell.

The composition of the vesica colony that comprises obvious enrichment can also comprise the vesica of specific size.For example, described vesica can all have and is greater than approximately 10,20 or the diameter of 30nm.They can all have the diameter of about 30-1000nm, about 30-800nm, about 30-200nm or about 30-100nm.In some embodiments, described vesica can all have and is less than approximately 10, the diameter of 000nm, 1000nm, 800nm, 500nm, 200nm, 100nm or 50nm.

For one or more features for the vesica colony of homogeneous can account for described composition total vesica colony at least about 30,40,50,60,70,80,90,95 or 99%.In some embodiments, in the biological sample that the composition of the vesica colony that comprises obvious enrichment is originated with described composition, the concentration of vesica is compared, the vesica concentration that the former comprises at least 2,3,4,5,10,20,25,50,100,250,500 or 1000 times.In other embodiment again, described composition can further comprise the vesica colony of the second enrichment, and wherein for one or more features as described herein, described vesica colony is at least 30% homogeneous.

Can obtain for for example, more than the obvious composition of enrichment of a kind of vesica colony (at least 2,3,4,5,6,7,8,9,10 kind of vesica colony) with multiple analysis.The vesica colony of each obvious enrichment can account at least 5,10,15,20,25,30,35,40,45,46,47,48 or 49% of the weight of described composition or quality.In some embodiments, obviously the vesica colony of enrichment account for the weight of described composition or quality at least about 30,40,50,60,70,80,90,95 or 99%.

Obviously the vesica colony of enrichment can be by obtaining by one or more methods disclosed herein, technique or system.For example, separate vesica colony and can implement by using for one or more wedding agents of one or more biomarkers of vesica in sample, for example use target in two or more wedding agents of two or more biomarkers of vesica.Can obtain with one or more trapping agents the vesica colony of obvious enrichment.Can identify by one or more detection reagent the vesica colony of obvious enrichment.

In one embodiment, the vesica colony that has a particular organisms marking is by using one or more wedding agents for the biomarker of the described biological marking to obtain.Can separate described vesica, thereby obtain comprising the composition of the vesica colony of the obvious enrichment with particular organisms marking.In another embodiment, have the biological marking of specific objective vesica colony can by use for and one or more wedding agents of the biomarker of the composition of the nontarget organism marking obtain.Therefore, described wedding agent can be for removing the vesica without target organism marking, and the composition of gained is obvious enrichment for the vesica colony with the biological marking of specific objective.The composition of gained can basically not exist comprise described wedding agent for the vesica of biomarker.

Detection system and test kit

The detection system of one or more biological markings that are configured to definite vesica also is provided.This detection system can be for detection of heterogeneous vesica colony or one or more homogeneous vesica colonies.Described detection system can be configured to detect multiple vesica, the biological marking that at least one subgroup of wherein said multiple vesica comprises another subgroup that is different from described multiple vesica.Described detection system detects at least 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds, 9 kinds, 10 kinds, 15 kinds, 20 kinds, 25 kinds, 30 kinds, 40 kinds, 50 kinds, 60 kinds, 70 kinds, 80 kinds, 90 kinds or 100 kinds of different vesica subgroups, and wherein each vesica subgroup comprises the different biological markings.For example, detection system (for example having used one or more methods disclosed herein, technique and composition) can be for detection of at least 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds, 9 kinds, 10 kinds, 15 kinds, 20 kinds, 25 kinds, 30 kinds, 40 kinds, 50 kinds, 60 kinds, 70 kinds, 80 kinds, 90 kinds or 100 kinds of different vesica colonies.

Described detection system can be configured to assess at least 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds, 9 kinds, 10 kinds, 15 kinds, 20 kinds, 25 kinds, 30 kinds, 40 kinds, 50 kinds, 60 kinds, 70 kinds, 80 kinds, 90 kinds, 100 kinds, 1000 kinds, 2500 kinds, 5000 kinds, 7500 kinds, 10 of one or more vesicas, 000 kind, 100,000 kind, 150,000 kind, 200,000 kind, 250,000 kind, 300,000 kind, 350,000 kind, 400,000 kinds, 450,000 kinds, 500,000 kind, 750,000 kind or 1,000,000 kind of different biomarker.In some embodiments, described one or more biomarkers are selected from Fig. 1,3-60, or biomarker as disclosed herein.Described detection system can be configured to assess specific vesica colony, for example, from the vesica in specific cells source; Perhaps, for assessment of multiple specific vesica colony, wherein each vesica colony has the specific biological marking.

Described detection system can be low density detection system or high-density detection system.For example, the low density detection system can detect the most nearly a kind, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds, 9 kinds or 10 kinds of different vesica colonies, and the high-density detection system can detect at least about 15 kinds, 20 kinds, 25 kinds, 50 kinds or 100 kinds of different vesica colonies.In another embodiment, the low density detection system can detect up to about 100 kinds, 200 kinds, 300 kinds, 400 kinds or 500 kinds of different biomarkers, and the high-density detection system can detect at least about 750 kinds, 1000 kinds, 2000 kinds, 3000 kinds, 4000 kinds, 5000 kinds, 6000 kinds, 7000 kinds, 8000 kinds, 9,000 kind, 10,000 kinds, 15,000 kinds, 20,000 kind, 25,000 kind, 50,000 kinds or 100,000 kinds of different biomarkers.In another embodiment again, the low density detection system can detect about 100 kinds, 200 kinds, 300 kinds, 400 kinds or the 500 kinds of different biological marking or biomarker combinations at the most, and the high-density detection system can detect at least about 750 kinds, 1000 kinds, 2000 kinds, 3000 kinds, 4000 kinds, 5000 kinds, 6000 kinds, 7000 kinds, 8000 kinds, 9,000 kind, 10,000 kind, 15,000 kind, 20,000 kind, 25,000 kind, 50,000 kind or 100,000 kinds of biological markings or biomarker combinations.

Described detection system can comprise optionally the probe with vesica hybridization.Described detection system can comprise the multiple probe for detection of vesica.In some embodiments, multiple probe is for detection of the amount of the vesica in heterogeneous vesica colony.In other embodiment again, multiple probe is for detection of the vesica colony of homogeneous.Multiple probe can for separating of or detect at least two kinds of different vesica subgroups, wherein each vesica subgroup comprises the different biological markings.

Detection system (for example having used one or more methods disclosed herein, technique and composition) can comprise multiple probe, these probe configuration for detection of or separate (for example using one or more methods disclosed herein, technique and composition) at least 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds, 9 kinds, 10 kinds, 15 kinds, 20 kinds, 25 kinds, 30 kinds, 40 kinds, 50 kinds, 60 kinds, 70 kinds, 80 kinds, 90 kinds or 100 kinds of different vesica subgroups, wherein each vesica subgroup comprises the different biological markings.

For example, detection system can comprise multiple probe, and these probes are arranged at least 2 kinds of detections, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds, 9 kinds, 10 kinds, 15 kinds, 20 kinds, 25 kinds, 30 kinds, 40 kinds, 50 kinds, 60 kinds, 70 kinds, 80 kinds, 90 kinds or 100 kinds of different vesica colonies.Described detection system can comprise multiple probe, these probes are arranged to optionally at least 2 kinds with one or more vesicas, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds, 9 kinds, 10 kinds, 15 kinds, 20 kinds, 25 kinds, 30 kinds, 40 kinds, 50 kinds, 60 kinds, 70 kinds, 80 kinds, 90 kinds, 100 kinds, 1000 kinds, 2500 kinds, 5000 kinds, 7500 kinds, 10, 000 kind, 100, 000 kind, 150, 000 kind, 200, 000 kind, 250, 000 kind, 300, 000 kind, 350, 000 kind, 400, 000 kind, 450, 000 kind, 500, 000 kind, 750, 000 kind or 1, 000, 000 kind of different biomarker hybridization.In some embodiments, described one or more biomarkers are selected from Fig. 1,3-60, or biomarker as disclosed herein.Described multiple probe can be arranged to the specific vesica of assessment colony, for example, from the vesica in specific cells source; Perhaps, for assessment of multiple specific vesica colony, wherein each vesica colony has the specific biological marking.

Described detection system can be for comprising low density detection system or the high-density detection system for detection of the probe of vesica.For example, the low density detection system can comprise the probe for detection of a kind at the most, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds, 9 kinds or 10 kinds different vesica colonies, and the high-density detection system can comprise the probe for detection of at least about 15 kinds, 20 kinds, 25 kinds, 50 kinds or 100 kinds different vesica colonies.In another embodiment, the low density detection system can comprise for detection of the about probe of 100 kinds, 200 kinds, 300 kinds, 400 kinds or 500 kinds different biomarkers at the most, and the high-density detection system can comprise for detection of at least about 750 kinds, 1000 kinds, 2000 kinds, 3000 kinds, 4000 kinds, 5000 kinds, 6000 kinds, 7000 kinds, 8000 kinds, 9,000 kind, 10,000 kind, 15,000 kind, 20,000 kind, 25,000 kind, 50, the probe of 000 kind or 100,000 kinds different biomarker.In another embodiment again, the low density detection system can comprise for detection of up to about 100 kinds, 200 kinds, 300 kinds, 400 kinds or 500 kinds of different biological markings or the probe of biomarker combinations, and the high-density detection system can comprise for detection of at least about 750 kinds, 1000 kinds, 2000 kinds, 3000 kinds, 4000 kinds, 5000 kinds, 6000 kinds, 7000 kinds, 8000 kinds, 9,000 kind, 10,000 kind, 15,000 kind, 20,000 kind, 25,000 kind, 50, the probe of 000 kind or 100,000 kinds of biological markings or biomarker combinations.

Described probe can be specifically for detection of specific vesica colony, for example there is the vesica of the particular organisms marking and vesica mentioned above.In addition, also provide the multiple probe for detection of the prostate specific vesica.Multiple probe can comprise the probe for detection of one or more following biomarkers: CD9, PSCA, TNFR, CD63, MFG-E8, EpCAM, Rab, CD81, STEAP, PCSA, 5T4, EpCAM, PSMA, CD59, CD66, CD24 and B7H3.Multiple probe for detection of Bcl-XL, ERCC1, keratin 15, CD81/TAPA-1, CD9, epithelial specific antigen (ESA) and mastocyte Chymotrypsin also is provided.

Multiple probe for detection of one or more miRNA of vesica can comprise the probe for detection of one or more following miRNA: miR-9, miR-629, miR-141, miR-671-3p, miR-491, miR-182, miR-125a-3p, miR-324-5p, miR-148b and miR-222.In another embodiment, described multiple probe comprises one or more probes for detection of EpCam, CD9, PCSA, CD63, CD81, PSMA, B7H3, PSCA, ICAM, STEAP and EGFR.In some embodiments, described multiple probe comprises one or more probes for detection of EpCam, CD9, PCSA, CD63, CD81, PSMA and B7H3.In other embodiments, described multiple probe comprises one or more probes for detection of EpCam, CD9, PCSA, CD63, CD81, PSMA, B7H3, PSCA, ICAM, STEAP and EGFR.In another embodiment again, a subgroup of described multiple probe is for one or more the trapping agent in EpCam, CD9, PCSA, CD63, CD81, PSMA, B7H3, PSCA, ICAM, STEAP and EGFR, and another subgroup is for detection of one or more in CD9, CD63 and CD81.Multiple probe also can comprise for detection of miR-92a-2 ^*, miR-147, miR-574-5p or its combination one or more probes.Multiple probe also can comprise one or more probes for detection of miR-548c-5p, miR-362-3p, miR-422a, miR-597, miR-429, miR-200a, miR-200b or its combination.Multiple probe also can comprise one or more probes for detection of EpCam, CK and CD45.In some embodiments, described one or more probes can be trapping agents.In another embodiment, described probe can be detection agent.In another embodiment again, described multiple probe comprises trapping agent and detection agent.

Described probe (such as trapping agent) can be connected with anchoring base, for example array or pearl.Perhaps, described probe (such as detection agent) is disconnected.Described detection system can be system or the system based on pearl of the system based on array, sequencing system, PCR-based, for example system mentioned above.Described detection system can be also microfluidic device mentioned above.

Described detection system can be the part of test kit.Perhaps, described test kit can comprise one or more probe groups or multiple probe, as described herein.Described test kit can comprise for example, probe for detection of vesica or multiple vesica (vesica in heterogeneous population).Described test kit can comprise the probe for detection of the vesica colony of homogeneous.For example, described test kit can comprise for detection of specific cells source vesica colony or have the probe of the vesica colony of the identical particular organisms marking.

Item Sets

Can set up the Item Sets of the multiplexing mark that instructs clinical decision and disease detection and management, sensitivity and the specificity of improvement are revealed in the combination of the biological marking in described Item Sets with respect to the single biological marking or the random biological marking combination table of selecting.In situation of the present invention, under the morbid state with respect to standard state, the multiple difference that can represent by the expression of the biological marking reflects the sensitivity of Item Sets.The specificity that can reflect in the dependency statistical measurement of for example, for example, signal with genetic expression (thering is target condition (standard deviation can be used as to this measuring)).In considering that biological marking group is included in to the process of Item Sets, the little standard deviation in measurement is relevant to higher specificity.In addition, other of variation measure (for example relation conefficient) also can be for this ability.

When in the present invention by biomarker or the combination of the biological marking, can apply the polynary index analysis of in-vitro diagnosis (IVDMIA) guide and rules.IVDMIA can be applied to be defined as by gene, gene alteration, sudden change, amplification, disappearance, polymorphism or methylate or the biological marking of the collection of two or more marks that any combination of the RNA that protein, peptide, polypeptide or RNA molecule, miRNA, mRNA, snoRNA, hnRNA maybe can combine forms, and makes the reasonable basis that is provided for making clinical correlated judgment (for example diagnosis, prognosis or treatment are selected) about the information that biological marking collection obtains in described group.These biological marking collection have formed each Item Sets of the present invention.Identical with most of diagnosis markers, use the mark of the minimum number be enough to make correct medical judgment normally desirable.This has prevented that the treatment of analyzing at products for further from incuring loss through delay and unsuitable time and resource use.Preferably, the set up item collection is so that the combination of the biological marking in described Item Sets shows sensitivity and the specificity of improvement for the single biological marking or the random biological marking combination of selecting.In situation of the present invention, under the morbid state with respect to standard state, the multiple difference that can show by the expression of the biological marking reflects the sensitivity of Item Sets.Specificity can be reflected in for example, dependency statistical measure to the signal of genetic expression and () target condition.Consider to be included in the mark group of the biological marking in Item Sets, any mathematical operations (it can show the higher sensitivity of generation, specificity, negative predictive value, positive predictive value or accuracy as a whole) that can be used from standard deviation, variance, covariance, relation conefficient, weighted mean, arithmetic sum, mean value, multiplication value, weighting or the equilibrium value of calculated value or scoring 2 kinds or multiple markers or these values also can be for this ability and also within the scope of the invention.

In another embodiment, can use mode identification method.An example relates to for various biomarkers (or biological marking Item Sets) and contrasts the express spectra of biomarker to be attributed to diagnosis.The express spectra that forms each biomarker of biological marking Item Sets is fixed in medium, for example computer-readable medium.

In an example, can set up form, wherein signal (for example intensity measurements) scope of input indication disease or physiological status.Then, can be by actual patient data and the value in form relatively, thus whether the sample of determining the patient is normal, optimum, ill or represents specific physiological status.In more complicated embodiment, record the pattern of expression signal (for example fluorescence intensity) with numeral or graphics mode.In the example of RNA, will compare from expression pattern and the described expression pattern of biological mark Item Sets (being combined with patient's sample) subsequently.Then, determine whether patient's sample has the pattern of the described disease of indication, the pattern, indication of indicating given prognosis are to the pattern of the possibility of the reaction for the treatment of or indicate the pattern of specific physiological status by the pattern comparison software.Then, the Item Sets of the express spectra of sample and compared with control cells is contrasted.If described sample expression pattern is consistent with the expression pattern of disease, prognosis or treatment correlated response, (in the situation that do not there is complementary medicine, consider) that the patient is diagnosed as the situation met with described these various environmental correclations.If described sample expression pattern is consistent with the expression pattern that is derived from normal/contrast vesica colony, for these situations, it is negative that the patient is diagnosed as.

In another exemplary, the method for setting up the biomarker expression Item Sets is by using optimized algorithm, for example widely used average variance algorithm in setting up the deposit Item Sets.This method is described in detail in the open No.20030194734 of U. S. application, and it is incorporated herein by reference.Perhaps, can use U.S. Patent No. 7,089,168,7,415,359 and the open No.20080208784,20040243354 and 20040088116 of U. S. application described in algorithm, system and method carry out modeling and analyze measured DNA to change, for the variation of mRNA, protein or the metabolite of the phenotype reading of effect and toxicity, each document all is incorporated to this paper with way of reference in full.

Biological marking Item Sets is selected and the example process of unknown sign is summarized as follows:

(1) select the baseline classification.

(2), for baseline classification sample, calculate mean value and the standard deviation of various biomarkers.

(3) calculate (the X* standard deviation+mean value) of each biomarker.This is that all other samples are by the baseline value of reading compared.X is the severity variable, and the X value that higher X value is lower is stricter.

(4) calculate the ratio of the baseline reading calculated in each laboratory sample and step 3.

(5) transformation ratio for example, so that be negative value (, using denary logarithm) lower than 1 ratio.(express that not enough biomarker suitably there is now MV optimize required negative value).

(6) ratio of these conversion is replied (asset returns) as input value for substituting the value of usually using in software application.

(7) described software is drawn effective border, and returns to the Item Sets of optimization at any some place along efficiency frontier.

(8) select required return of value or the variance on efficiency frontier.

(9) by adding and the intensity level of each gene and the long-pending value of calculating each sample Item Sets of weight, weight is produced by the Item Sets selection algorithm.

(10) by the long-pending phase Calais computation bound value with the standard deviation of Y and the biological marking Item Sets of described baseline value by the average biological marking Item Sets value of baseline group.The value that is greater than this cut off value should be classified as the experiment classification.

(11) optionally, can repeat this process until optimum prediction.

In addition, select biological marking Item Sets can also comprise the application of heuristic rule.Preferably, this rule is based on biology and the understanding of the technology for generation of clinical effectiveness is planned.More preferably, they are applied to the output of self-organization method.For example, for the multiple biomarker at differential expression in having the experimenter of specified disease, the average variance method that biological marking Item Sets is selected can be applied to microarray data.Described method is output as the optimization group of the biomarker that can be included in those biomarkers of expressing in vesica and diseased tissue.If for the sample of testing method derive from vesica and in the situation of disease or physiological status some biomarker of differential expression in vesica, be also differential expression, can use heuristic rule, wherein biological marking Item Sets is selected from the efficiency frontier of having got rid of differential expression in vesica.Certainly, can before forming efficiency frontier, apply described rule (for example, by the described rule of application in data preliminary election process).

For analysis, feature extraction and the signal deconvolution of carrying out the linearity of extensive data set and nonlinear characteristic subspace identify that the multiple analysis thing in vesica source is composed to be diagnosed, prognosis and treatment select and/or determine that other statistics, mathematics and the computerized algorithm of the sign of physiological status can implement with any combination without the supervision analytical procedure, include but not limited to: principle component analysis (PCA) and linear and non-linear independent component analysis (ICA); The separation of blind source, non-Gauss analysis, the assessment of natural gradient PRML; Joint approximate diagonalization; Eigenmatrix; The continuous floating forward direction of Gaussian radial basis function, kernel and multinomial Kernel analyze is selected.

Computer system

Can analyze vesica for characterization of molecules, for example for example, amount, existence by measuring one or more biomarkers (listed in Fig. 1,3-60) or do not exist.The data that generate can be for generation of the biological marking, and it can store and analyze by computer system, for example, shown in Figure 65.In addition, analyze the biological marking or the biological marking and one or more phenotypic correlations connection also can be implemented by computer system to (but for example by using the computer actuating logic).

Computer system (for example, shown in Figure 65) can be for transmitting data and result after analysis.Therefore, Figure 65 is the sketch that shows the representative illustration logical device, by this equipment, can analyze from the result of vesica and report or produce described analysis.Figure 65 shows computer system (or digital device) 800 to receive or to store the data that produced by vesica, to analyze the report of described data to produce one or more biological markings and to produce one or more biological markings (or phenotype sign).In addition, described computer system can also be implemented relatively and analyze the produced biological marking, and transmits the result of gained.Perhaps, described computer system can be accepted the raw data (for example passing through network transmission data) that vesica is analyzed, and is analyzed.

Computer system 800 can be understood as can be from the logical device of medium 811 and/or the network port 805 reading command, and it can optionally be connected with the server 809 with mounting medium 812.System shown in Figure 65 comprises CPU 801, disc driver 803, optional input unit such as keyboard 815 and/or mouse 816, and optional watch-dog 807.With the data corresponding of the server 809 of local or far away and position can by shown in telecommunication media realize.Described telecommunication media can comprise any means that transmit and/or receive data.For example, telecommunication media can connect for network connection, wireless connections or internet.This connection can provide communication on World Wide Web.Can imagine, can or connect at these networks and be transmitted about data of the present invention, in order to received and/or check by certain side 822.Take over party 822 can be but be not limited to individuality, health care supplier or health care management person.Therefore, about the information of test result and data can this world generation Anywhere and be transferred into different location.For example, when the buildings different, city, state, country, continent or while abroad being analyzed, the information of test result and data can and form transmissible form according to generation mentioned above.Therefore, but the test result of delivery form can be transfused to the U.S. to arrive take over party 822.Therefore, but the information for generation of the delivery form of the diagnosis of one or more samples for from individuality has also been contained in the present invention.The step that described method comprises has: (1) the method according to this invention is determined diagnosis, prognosis or treatment diagnosis etc. by described sample; And (2) but the result of described determining step is presented as to delivery form.But described delivery form is the product of described production method.In one embodiment, computer-readable medium comprises the medium of the result (the biological example marking) that is applicable to transmit biological sample analysis.Described medium can comprise the result (for example experimenter's the biological marking) that relates to vesica, and wherein said result is used method as herein described to obtain.

The external collection of vesica

In addition, can also be from vitro collection for the vesica of analysis and definite phenotype.Can be cultivated cell, thereby the vesica that the target cell in culture discharges can be spontaneously to obtain or can be stimulated vesica is discharged in substratum.(such as referring to 1998.Nat.Med.4:594-600 such as Zitvogel; The 2004.J.Immunol.172:2137-214631:2892-2900 such as Chaput; The 2005.J.Transl.Med.3:10 such as Escudier; Morse etc. 2005, J.Transl.Med.3:9; The 2006.Am.J.Transplant.6:1541-1550 such as Peche; The 2005.J.Immunol.174:6440-6448 such as Kim, all these documents are incorporated herein by reference in full).Can clone or tissue sample grow to 80% converge after, in fresh DMEM, cultivate 72 hours.Can stimulate subsequently vesica to generate (process such as the heat-shocked referring to described melanoma cell of 2003.Cancer Res.63:8212-8220 such as Dressel, it is incorporated herein by reference in full).Then, can gather in the crops supernatant liquor, and according to the vesica for preparing described herein.

In an example, the in vitro vesica produced can be cultivated and be obtained by target cell source or clone, and vesica can separate material, semiconductor nanocrystal or other nano particle (comprising in vivo as quantum dot or the gold grain again introduced for the marker of imaging analysis) that obtain and use subsequently magnetic mark thing, fluorescence part, radio isotope, enzyme, chemical reflective probe, metallic particles, nonmetal colloidal particle, polymeric dye particles, pigment molecule, granules of pigments, electrochemical activity by cell culture medium and carry out mark.Can be by setting up for example, culture condition for the given cell source with target signature (lung carcinoma cell or culture with clone of known EGFR sudden change, wherein said sudden change has been given the resistance of Gefitinib or susceptibility); Then described cell culture is exposed to Gefitinib; Separation is by the vesica of described culture generation and finding on array that analyzing these vesicas uses the vesica of isolated culture alternatively to identify the novel biological marking to find novel antigens or the wedding agent (this antigen or wedding agent can be used as the biological marking to catch the vesica of this kind) of expressing in the vesica outside subsequently.In addition, likely be separated in any other biomarker of finding in these vesicas or the biological marking with for finding the novel marking (including but not limited to nucleic acid, protein, lipid or their combination) that can there is clinical diagnosis, prognosis or treat relevant prompting.

Also can be at first from destination organization separate targets cell being cultivated.For example, can use sterile equipment and plastic ware by patient's scalp, pull up individually in growth phase (anagen) the hair follicle of human hair, note not damaging hair follicle.Each sample can be transferred to the petri diss be equipped with for the aseptic PBS of tissue culture.The hair follicles of the human hair early growth period of separation can be transferred to carefully in the single hole of the 24 hole flat boards that 1ml William ' s E substratum is housed.Hair follicle can be remained on to 37 ℃, 5%CO in the mode of unmanaged flexibility ₂in humidification incubator in the atmosphere of 95% air.Can within every 3 days, change substratum, note not damaging hair follicle.Then, collecting cell, and centrifugal in substratum.Then, described method before can using, use the specific vesica of described cell source had to specific antigen or the Cell binding companion body separates vesica.Then can separate and identification of organism mark and the biological marking by method known to those of skill in the art.

Can also be under microgravity or below-G conditions or under the environment of free-falling the training objective cell.For example, the NASA bioreactor technology can make described cell with faster speed and much bigger quantity growth.Then, described method before can using, use the specific vesica of described cell source had to specific antigen or the Cell binding companion body separates vesica.

Rotation wall container or RWVersus are a class by the NASA exploitation and for the bio-reactor of NASA, and it is designed to the suspension culture of grown cell under the static environment that has imitated microgravity.(for example, referring to U.S. Patent No. 5,026,650; 5,153,131; 5,153,133; 5,437,998; 5,665,594; 5,702,941; 7,351,584,5,523,228,5,104,802,6,117,674; Martin etc., Trends in biotechnology 2004; 22; 80-86, Li etc., Biochemical Engineering Journal 2004; 18; 97-104, Ashammakhi etc., Journal Nanoscience Nanotechnology 2006; 9-10:2693-2711, Zhang etc., International Journal of Medicine 2007; 4:623-638, Cowger, N L etc., Biotechnol.Bioeng 64:14-26,1999, Spaulding, G F etc., J.Cell.Biochem.51:249-251,1993, Goodwin, T J etc., Proc.Soc.Exp.Biol.Med.202:181-192,1993; Freed, LE etc., In Vitro Cell.Dev.Biol.33:381-385,1997, Clejan, S. etc., Biotechnol.Bioeng.50:587-597,1996) .Khaoustov, VI etc., In Vitro Cell.Dev.Biol.35:501-509.1999, each document all is incorporated herein by reference in full).

Perhaps, can be according in U.S. Patent No. 6,911,201 and the open No.20050181504,20050180958,20050176143 and 20050176137 of U. S. application in the method usually described, cultivate separated target cell or the specific vesica of cell source in stationary phase piston flow bio-reactor, each document all is incorporated herein by reference in full.Perhaps, can also be according in U.S. Patent No. 5,486, the method for general description is separated and training objective cell or the specific vesica of cell source in 359.

As U.S. Patent No. 5,486,359 (they are incorporated herein by reference in full) are usually described, and an embodiment can comprise the following steps: the tissue samples that comprises target cell or cell source specificity vesica is provided; To join in substratum from cell or the vesica of this tissue samples, it only allows target cell or the specific vesica of cell source optionally to adhere on substrate surface when being cultivated; Culture sample-substratum mixture; And by removing the material do not adhered on substrate surface.

Embodiment

embodiment 1: vesica is from the purifying of prostate cancer cell line

In the substratum that comprises 20%FBS (foetal calf serum) and 1%P/S/G, prostate cancer cell line is cultivated to 3-4 days.Then under 4 ℃, by cell under 400 * g pre-centrifugal 10 minutes.Retain supernatant liquor, and under 4 ℃ under 2000 * g centrifugal 20 minutes.Can use the concentrated supernatant liquor that comprises vesica of Millipore Centricon Plus-70 (numbering UFC710008 Fisher).

At room temperature, with the PBS of 30ml with 1000 * g by centrifugal ultrafiltration pipe pre-wash 3 minutes.Then, the pre-eccentric cell culture supernatants of 15-70ml is poured in concentrated cup (Concentrate Cup), and at room temperature in Swing Bucket Adapter (Fisher numbers 75-008-144) with 1000 * g centrifugal 30 minutes.

Percolation thing in collection cups (Collection Cup) is poured out.Use extra supernatant liquor arbitrarily that the volume in described concentrated cup is adjusted to 60ml.At room temperature by described concentrated cup with centrifugal 30 minutes of 1000 * g with the concentrating cells supernatant liquor.

By adding the described concentrated cup of 70ml PBS washing, and under 1000 * g centrifugal 30-60 minute until remain about 2ml.By enriched material being inverted in little sample cup and removing vesica from strainer in centrifugal 1 minute under 4 ℃.Use PBS that volume is adjusted to 25ml.Vesica this moment concentrates, and is added on 30% sucrose pad.

In order to make pad, by 4ml Tris/30% sucrose/D2O solution, (30g is without sucrose, 2.4g Tris alkali, the 50ml D2O of proteolytic enzyme, drip 10N NCL by pH regulator to 7.4, use D2O by volume-adjustment to 100ml, carry out sterilizing by the 0.22um strainer) be loaded into the bottom of thin-walled ultracentrifugation pipe at the bottom of the 30ml V-type.Above described sucrose pad, in the situation that not disturbance interface gently adds the 25ml concentrating vesicles of dilution, and under 4 ℃ with 100,000 * g centrifugal 75 minutes.Use the 10ml transfer pipet remove carefully sucrose pad top～25ml, and, with the vesica of apicule transfer pipet (SAMCO 233) collection～3.5ml, then be transferred in new ultracentrifugation pipe, wherein be added with 30ml PBS.Under 4 ℃ with 100,000 * g by centrifugal 70 minutes of described pipe.Pour out carefully supernatant liquor.Agglomerate is resuspended in 200ul PBS, and can be stored under 4 ℃ or for analyzing.BCA analytical method (1: 2) can be for measuring the content of protein, and Western trace or electron microscopy can be for determining the purifying of vesica.

embodiment 2: vesica is from the purifying of VCaP and 22Rv1

By at first using isopyknic PBS (1ml) diluting plasma, collect the vesica from prostate gland Vertebral Neoplasms: (VCaP) and 22Rv1 (it is the PC-3, is derived from human prostata cancer heterograft (CWR22R)) by ultracentrifugation.By the fluid transfer of dilution to 15ml Falcon centrifuge tube, and under 4 ℃ with 2000 * g centrifugal 30 minutes.Supernatant liquor (～2ml) is transferred in ultracentrifugation pipe 5.0ml PA thin-walled tube (Sorvall#03127), and under 4 ℃ with 12000 * g centrifugal 45 minutes.

Supernatant liquor (～2ml) is transferred in new 5.0ml ultracentrifugation pipe, and adds 2.5ml PBS to be filled to maximum volume, then under 4 ℃ with 110,000 * g centrifugal 90 minutes.Pour out supernatant liquor and do not upset agglomerate, this agglomerate is resuspended in 1ml PBS.Add 4.5ml PBS and described pipe is filled to maximum volume, and under 4 ℃ under 110,000 * g centrifugal 70 minutes.

Pour out supernatant liquor and do not confuse agglomerate, and add other 1ml PBS with the washing agglomerate.Add 4.5ml PBS and volume is increased to maximum volume, and under 4 ℃ with 110,000 * g centrifugal 70 minutes.Use the P-1000 transfer pipet to remove supernatant liquor, until be the PBS of～100 μ l at the bottom of described pipe.Use remove～90 μ l residues of P-200 transfer pipet, and gently be drawn in micro-centrifuge tube by the agglomerate that uses will the use～10 μ l PBS residues collections of P-20 transfer pipet.Use the fresh PBS of other 5 μ l to wash out residual agglomerate from the bottom of drying tube, and collect in micro-centrifuge tube, and be suspended in phosphate buffer salt (PBS) to concentration be 500 μ g/ml.

embodiment 3: the collection of blood plasma and the purifying of vesica

Puncture by standard collects blood in 7ml K2-EDTA pipe.In the whizzers of 4 ℃ (SORVALL Legend RT+ whizzer), with 400g by centrifugal 10 minutes of this sample, thereby isolate blood plasma in hemocyte.By drawing carefully, supernatant liquor (blood plasma) is transferred in 15ml Falcon centrifuge tube.By centrifugal 20 minutes of blood plasma, and collect supernatant liquor under 2,000g.

For storage, the blood plasma of about 1ml (supernatant liquor) is distributed in freeze pipe, be positioned in dry ice so that they freeze, and be stored under-80 ℃.Before the purifying vesica, if sample is stored under-80 ℃, sample 5 minutes thaws in cooling bath.Manually described sample is put upside down to mixing, thereby insoluble substance is dissipated.

For the first time pre-centrifugal in, use isopyknic PBS (for example, using 2ml PBS to dilute the blood plasma of about 2ml) diluting plasma.Diluent is transferred in 15ml Falcon pipe, and under 4 ℃ with 2000 * g centrifugal 30 minutes.

For centrifugal in advance for the second time, supernatant liquor (approximately 4ml) is transferred in 50ml Falcon pipe carefully, and in the Sorval whizzer under 4 ℃ with 12,000 * g centrifugal 45 minutes.

In separating step, use the P1000 transfer pipet that supernatant liquor (approximately 2ml) is transferred in 5.0ml ultracentrifugation PA thin-walled tube (Sorvall#03127) carefully, and use other 0.5ml PBS to be filled to maximum volume.Under 4 ℃ by described pipe with 110,000 * g centrifugal 90 minutes.

In washing for the first time, pour out supernatant liquor and do not upset agglomerate.With 1ml PBS, by this agglomerate resuspension or washing, and use other 4.5ml PBS that described pipe is filled to maximum volume.Under 4 ℃ by described pipe with 110,000 * g centrifugal 70 minutes.By repeating identical step, wash for the second time.

By with the P-1000 transfer pipet, removing supernatant liquor until be that about 100 μ l PBS collect vesica at the bottom of described pipe.Remove about 90 μ l PBS and abandon with the P-200 transfer pipet.By gently draw to collect agglomerate and remaining PBS with the P-20 transfer pipet.Use the fresh PBS of other 5 μ l to wash out residual agglomerate from the bottom of described drying tube, and collect in micro-centrifuge tube.

embodiment 4: with the microballoon of antibody coupling and the antibody of directly coupling, analyze vesica

The present embodiment has been showed the use with the particulate of antibody coupling, the described vesica of wherein said antibody capture.For example,, referring to Figure 63 A.Antibody (detection antibody) and the direct coupling of marker, and for detection of the biomarker of catching on vesica.

At first, select the microsphere set (Luminex, Austin, TX) of antibody coupling.Described microsphere set can comprise various antibody, and therefore can carry out multiplexing.Mix supersound process by vortex and within about 20 seconds, make described microballoon resuspension.Carry out the preparation work mixture of microspheres by the microballoon stoste of coupling being diluted to final concentration in Startblock (Pierce (37538)) for 100 microballoon/μ L of each collection.50 μ L work mixture of microspheres are for each hole.PBS-1%BSA or PBS-BN (0.05% trinitride, pH 7.4 for PBS, 1%BSA) can be with the buffer reagents that performs an analysis.

1.2 μ m Millipore filter plates are wetting in advance with the PBS-1%BSA (Sigma (P3688-10PAK+0.05% sodiumazide (S8032))) in 100 μ l/ holes, and draw by vacuum manifold (vacuum manifold).The described work mixture of microspheres of 50 μ l equal portions is allocated in the suitable hole of this filter plate (Millipore Multiscreen HTS (MSBVN1250)).The standard substance of 50 μ l equal portions or sample are assigned in suitable hole.Cover this filter plate, and at room temperature hatch 60 minutes on plate vibrator.Cover described filter plate with sealing, be placed on the whirling vibration device, and be set in 900 times lasting 15-30 seconds so that described pearl resuspension.Afterwards, the time that Speed Setting is being continued to hatch for 550 times.

By vacuum manifold, supernatant liquor is drawn to (in all absorption steps all lower than 5 inches of mercury).Each hole is washed to 2 times with the PBS-1%BSA (Sigma (P3688-10PAK+0.05% sodiumazide (S8032))) of 100 μ l, and draw by vacuum manifold.Microballoon is resuspended in the PBS-1%BSA (Sigma (P3688-10PAK+0.05% sodiumazide (S8032))) of 50 μ L.The detection antibody of PE coupling is diluted to 4 μ g/mL (or suitable concentration) in PBS-1%BSA (Sigma (P3688-10PAK+0.05% sodiumazide (S8032))).(annotate: the dilution of each reaction needed 50 μ L detects antibody.) the diluted detection antibody of 50 μ l equal portions is joined in each hole.Cover filter plate, and at room temperature hatch on plate vibrator 60 minutes.Cover described filter plate with sealer, be placed on the whirling vibration device, and be set in 900 times lasting 15-30 seconds so that described pearl resuspension.Afterwards, the time that Speed Setting is being continued to hatch for 550 times.By vacuum manifold, supernatant liquor is drawn.Each hole is washed to 2 times with the PBS-1%BSA (Sigma (P3688-10PAK+0.05% sodiumazide (S8032))) of 100 μ l, and draw by vacuum manifold.Microballoon is resuspended in the PBS-1%BSA (Sigma (P3688-10PAK+0.05% sodiumazide (S8032))) of 100 μ L.Analyze described microballoon according to the guide of described system on the Luminex analyser.

Figure 66 has described the microballoon that antibody is puted together, and is combined with the microballoon of the antibody coupling of vesica on it.Particularly, the described scanning electron photomicrograph (SEM) that illustrates the coupling pearl of puting together with EpCam, wherein said pearl hatches together with the VCaP vesica.For the figure shown in Figure 66 A, slide is used poly-l-lysine coating slide and is hatched together with described pearl solution.After connection, described pearl (i) is used glutaraldehyde and the fixing described pearl (i) of perosmic anhydride successively, and each fixing step is 30 minutes, and washing a little between two fixing step; (ii) dehydration gradually in acetone, wherein said acetone increases progressively with 20%, the about 5-7 minute of each step; (iii) carrying out critical point drying; And (iv) with golden splash coating.Figure 66 B, left side described on the pearl of the EpCam as shown in Figure 66 A coating than the vesica of high-amplification-factor.Figure 66 B, right side has been described by ultracentrifugation and has been separated, and is attached on the slide glass that is coated with the poly-L-Lysine coating and according to the vesica of fixing and dyeing described in Figure 66 A.

embodiment 5: with microballoon and the biotinylated antibody of antibody coupling, analyze vesica

The present embodiment has been showed the use with the particulate of antibody coupling, the described vesica of wherein said antibody capture.Antibody (detection antibody) bioid.With the marker of streptavidin coupling for detection of biomarker.

At first, select the microsphere set (Luminex, Austin, TX) of suitable antibody coupling.Within about 20 seconds, make described microballoon resuspension by vortex and supersound process.Carry out the preparation work mixture of microspheres by the microballoon stoste of this coupling being diluted to final concentration in Startblock (Pierce (37538)) for 50 microballoon/μ L of each collection.(annotate: need 50 μ L work mixture of microspheres in each hole.) pearl in Start Block should seal 30 minutes and be no more than 1 hour.

By the PBS-1%BSA+ trinitride (PBS-BN) in 100 μ l/ holes for 1.2 μ m Millipore filter plates, ((Sigma (P3688-10PAK+0.05% sodiumazide (S8032))) is wetting in advance, and draws by vacuum manifold.The described work mixture of microspheres of 50 μ l equal portions is allocated in the suitable hole of this filter plate (Millipore Multiscreen HTS (MSBVN1250)).The standard substance of 50 μ l equal portions or sample are assigned in suitable hole.Cover this filter plate with sealer, and at room temperature hatch on the filter plate vibrator 60 minutes.The filter plate of covering is placed on the whirling vibration device, and is set in 900 times lasting 15-30 seconds with the described pearl of resuspension.Afterwards, the time that Speed Setting is being continued to hatch for 550 times.

By vacuum manifold, supernatant liquor is drawn to (in all absorption steps all lower than 5 inches of mercury).Can use the Pall vacuum manifold to be drawn.When being placed on described manifold by this filter plate, valve being placed on fully and closing (full off) position.In order to draw lentamente, the valve opening, with draw fluid from described hole, for the sample by 100 μ l and pearl sucking-off in hole fully, is needed to about 3 seconds.Once described sample is drained, press the purge button (purge button) on manifold, thereby discharge residual vacuum pressure from filter plate.

Each hole is washed to 2 times with the PBS-1%BSA+ trinitride (PBS-BN) (Sigma (P3688-10PAK+0.05% sodiumazide (S8032))) of 100 μ l, and draw by vacuum manifold.Described microballoon is resuspended to the PBS-1%BSA+ trinitride (PBS-BN) of 50 μ L (in (Sigma (P3688-10PAK+0.05% sodiumazide (S8032))).

Biotinylated detection antibody is diluted to 4 μ g/mL in PBS-1%BSA+ trinitride (PBS-BN) (Sigma (P3688-10PAK+0.05% sodiumazide (S8032))).(annotate: the detection antibody of each reaction needed 50 μ L dilutions.) the diluted detection antibody of 50 μ l equal portions is joined in each hole.

Cover this filter plate with sealer, and at room temperature hatch on the plate vibrator 60 minutes.Described plate is placed on to orbit determination vibration upper, and is set in lasting 15-30 second 900 times, thus the described pearl of resuspension.Afterwards, within the time of hatching by Speed Setting at 550 times.

By vacuum manifold, supernatant liquor is drawn.Can use the Pall vacuum manifold to complete absorption.When being placed on described manifold by this plate, valve being placed in fully and closing (full off) position.In order to draw lentamente, the valve opening, with draw fluid from hole, for the sample by 100 μ l and pearl sucking-off in hole fully, is needed to about 3 seconds.Once all samples drain, just press and empty button (purge button) on manifold, thereby discharge residual vacuum pressure from this plate.

Each hole is washed to 2 times with the PBS-1%BSA+ trinitride (PBS-BN) (Sigma (P3688-10PAK+0.05% sodiumazide (S8032))) of 100 μ l, and draw by vacuum manifold.Described microballoon is resuspended in the PBS-1%BSA (Sigma (P3688-10PAK+0.05% sodiumazide (S8032))) of 50 μ l.

Streptavidin-R-PE acceptor (molecular probe 1mg/ml) is diluted to 4 μ g/mL in PBS-1%BSA+ trinitride (PBS-BN).Each reaction is used to the streptavidin-R-PE of 50 μ l dilutions.Diluted streptavidin-the R-PE of 50 μ l equal portions is joined in each hole.

Cover this filter plate with sealer, and at room temperature hatch on the plate vibrator 60 minutes.Described plate is placed on orbital shaker, and is set in lasting 15-30 second 900 times, thus the described pearl of resuspension.Afterwards, within the time of hatching by Speed Setting at 550 times.

By vacuum manifold, supernatant liquor is drawn.Can use the Pall vacuum manifold to complete absorption.When being placed on described manifold by this plate, valve is placed in to complete off-position place.In order to draw lentamente, the valve opening, with draw fluid from hole, for the sample by 100 μ l and pearl sucking-off in hole fully, is needed to about 3 seconds.Once all sample drains, and just presses the button that empties on manifold, thereby discharge residual vacuum pressure from this plate.

Each hole is washed to 2 times with the PBS-1%BSA+ trinitride (PBS-BN) (Sigma (P3688-10PAK+0.05% sodiumazide (S8032))) of 100 μ l, and draw by vacuum manifold.Described microballoon is resuspended in the PBS-1%BSA+ trinitride (PBS-BN) (Sigma (P3688-10PAK+0.05% sodiumazide (S8032))) of 100 μ l, and is analyzed on the Luminex analyser according to described system guide.

embodiment 6: antibody purification and with the carbodiimide coupling of carboxylation microballoon

Antibody purification scheme: use protein G resin (Pierce is Thermo Fisher Scientific, Rockford, the part of IL for Protein G spin kit, the production number 89979) antibody purification from Pierce.Miniature chromatographic column that will be prepared by the P-200 suction nozzle filtered is for purifying.

Use is loaded on the protein G resin of 100 μ l in each micro-column from 100 μ l damping fluids of described Pierce test kit.After waiting and within several minutes, making described resin settled, use when needed P-200 pipettor (Pipettman) to apply air pressure to drain damping fluid, and guarantee that this post can be dry.(pH 7.4,100mM phosphate buffered saline buffer, 150mM NaCl for the binding buffer liquid of use 0.6ml; (Pierce, production number 89979)) this post of balance.Antibody is applied to this post (antibody of<1mg is loaded on this post).Use 1.5ml binding buffer liquid to wash this post.Prepared 5 pipes (1.5ml micro-centrifuge tube) and 10 μ l neutralization solutions (Pierce, production number 89979) have been applied to each pipe.Use from the elution buffer of described test kit by antibody elution to each pipe of described 5 pipes, every pipe 100ul (amounting to 500 μ l).Use Nanodrop (Nanodrop 1000 spectrophotometers, Nanodrop is Thermo Scientific, Wilmington, the part of DE) to measure the relative absorbancy of each fraction under 280nm.Selection has the fraction of the highest OD reading and uses for downstream.Use Pierce Slide-A-Lyzer Dialysis Cassette (Pierce, production number 66333,3KDa cutoff) with 0.25 liter of PBS damping fluid described sample of dialysing.With every 2 hours exchange buffering liquid of continuous stirring, minimumly carry out three times and change under 4 ℃.Subsequently described dialysis sample is transferred in the 1.5ml micro-centrifuge tube, and can is identified and be stored under 4 ℃ (short-terms) or-20 ℃ (for a long time).

coupling: according to following scheme, use above listed corresponding antibody separately to apply microballoon.

Should be protected in order to avoid be exposed to for a long time under light at microballoon described in this process.According to described microballoon (xMAP technologies, MicroPlex ^tMmicrospheres, Luminex Corporation, Austin, TX) the coupling raw material microballoon not of the indication resuspension described in the product information page that provides.By 5 * 10 ⁶individual raw material microballoon is transferred in USA Scientific 1.5ml micro-centrifuge tube (USA Scientific, Inc., Orlando, FL).By at room temperature with >=8000 * g, carrying out the micro-centrifugal raw material microballoon that precipitates of 1-2 minute.Remove supernatant liquor and approximately 20 seconds the microballoon of precipitation was resuspended to the dH of 100 μ l by vortex and supersound process ₂in O.By at room temperature with >=8000 * g, carrying out the micro-centrifugal microballoon that precipitates of 1-2 minute.Remove supernatant liquor and approximately the microballoon through cleaning is resuspended to the 100mM SODIUM PHOSPHATE, MONOBASIC of 80 μ l 20 seconds by vortex and supersound process (Branson 1510, Branson Ultrasonics Corp.Danbury, CT), in pH 6.2.The 50mg/ml Sulfo-NHS of 10 μ l (Thermo Scientific, number 24500) (is diluted in to dH ₂in O) add to described microballoon and gently mix by vortex.The 50mg/ml EDC of 10 μ l (Thermo Scientific, numbering 25952-53-8) (is diluted in to dH ₂in O) add to described microballoon and gently mix by vortex.At room temperature hatch described microballoon 20 minutes and gently mix by vortex with the interval of 10 minutes.By at room temperature with >=8000 * g, carrying out the micro-centrifugal microballoon activated that precipitates of 1-2 minute.Remove supernatant liquor and approximately 20 seconds described microballoon was resuspended to the 50mM MES of 250 μ l by vortex and supersound process, in pH 5.0 (MES, Sigma, numbering M2933, Sigma-Aldrich, St.Louis, MO).Only ((Sigma (P3688-10PAK+0.05% sodiumazide (S8032))) should be used as analysis buffer and lavation buffer solution PBS-1%BSA+ trinitride (PBS-BN).Precipitate described microballoon by room temperature with >=8000 * g, carrying out 1-2 minute micro-centrifugal subsequently.

Remove supernatant liquor and approximately 20 seconds described microballoon was resuspended to the 50mM MES of 250 μ l by vortex and supersound process, in pH 5.0 (MES, Sigma, numbering M2933).Only ((Sigma (P3688-10PAK+0.05% sodiumazide (S8032))) should be used as analysis buffer and lavation buffer solution PBS-1%BSA+ trinitride (PBS-BN).Precipitate described microballoon by room temperature with >=8000 * g, carrying out 1-2 minute micro-centrifugal subsequently, complete therefrom 50mM MES, twice washing that pH 5.0 carries out.

The microballoon of removing supernatant liquor and approximately will activating and wash 20 seconds by vortex and supersound process is resuspended to the 50mM MES of 100 μ l, in pH 5.0.The protein of 125,25,5 or 1 μ g amount is added to the microballoon of resuspension.(annotate: can carry out titration to measure the optimum protein quality of each concrete linked reaction in 1 to 125 μ g scope.) using 50mM MES, pH 5.0 is adjusted to 500 μ l by cumulative volume.At room temperature mix by vortex mixed linked reaction thing and by it under (by Labquake rotator, Barnstead, be rotated on Thermo Scientific) and hatch 2 hours.By at room temperature with >=8000 * g, carrying out the micro-centrifugal microballoon that precipitates coupling of 1-2 minute.Remove supernatant liquor and approximately 20 seconds the microballoon of described precipitation be resuspended in the PBS-TBN of 500 μ l by vortex mixed and supersound process.(can optimize concentration for the concrete reagent used, analysis condition, multiplexing level etc.)

Described microballoon at room temperature follows mixing (by Labquake rotator, being rotated on Barnstead) to hatch 30 minutes.By at room temperature with >=8000 * g, carrying out the micro-centrifugal microballoon that precipitates coupling of 1-2 minute.Remove supernatant liquor and approximately 20 seconds described microballoon be resuspended in the PBS-TBN of 1ml by vortex and supersound process.(carry out sample at every turn, when detecting antibody or SA-PE and adding, use sealer and shade (such as aluminium foil) to cover described flat board, be placed on the whirling vibration device, and be set in 900 times lasting 15-30 seconds with the described pearl of resuspension.The time that afterwards, Speed Setting should continued to hatch for 550 times).

Micro-centrifugally precipitate described microballoon by what carry out 1-2 minute with >=8000 * g.Remove described supernatant liquor and approximately 20 seconds described microballoon be resuspended in the PBS-TBN of 1ml by vortex and supersound process.By carry out the micro-centrifugal described microballoon (obtaining twice washing of total of using 1ml PBS-TBN to carry out) that precipitates of 1-2 minute with >=8000 * g.

embodiment 7: it is concentrated that vesica carries out from blood plasma

Equipment and equipment: Pall life sciences Acrodisc, 25mm syringe filter w/1.2um, Versapor film (aseptic), unit number: 4190; Pierce thickener 7ml/150K MWCO (weight shutoff value), unit number: 89922; The BD syringe filter, 10ml, unit number: 305482; Sorvall Legend RT Plus series desktop whizzer, have the 15ml swinging bucket rotor; PBS, pH 7.4, Sigma P3813-10PAK, it is prepared in the water of aseptic molecular level; Copolymer 1 .7ml micro-centrifuge tube, USA Scientific, numbering 1415-2500.Water for reagent is the water (Sigma, numbering W4502) of the molecular level of sterile filtration.Operating in Biohazard Safety Equipment of patient's blood plasma carried out.

Program technic:

1. to the filtration procedure of plasma sample

1.1. take out plasma sample from-80 ℃ of (65 ℃ to-85 ℃) refrigerators.

The sample (10-15 minute) 1.2. thaw in the water of room temperature.

1.3. prepare syringe and filter by from its packing, taking out necessary amount.

1.4. pull inner core so that the aseptic molecular level water of 4mL is sucked in described syringe.The filter of 1.2 μ m is attached to the top of syringe and makes content pass through described filter and arrive on described 7ml/150K MWCO Pierce post.

1.5. cover described post and be placed in described bucket centrifuge to descend centrifugal 4 minutes at 20 ℃ (16 ℃-24 ℃) at Sorvall Legend RT plus whizzer with 1000 * g.

1.6. when carrying out when centrifugal, this filter being pulled down from syringe.Subsequently inner core is taken out from syringe.

1.7. abandon the percolation liquid of pipe and gently on paper handkerchief the tapped post to remove the moisture of any remnants.

1.8. measure and record the original volume of all plasma samples.Have lower than the sample of 900 μ l volumes and may do not processed.

1.9. will open syringe and filter is placed on open Pierce post.Opening end at syringe fills with 1 * PBS of 5.2mL and with pipettor, blood plasma is sneaked into to PBS tri-to four times.

1.10. insert once again plunger and oppress lentamente this inner core until the content of described syringe has entered on described Pierce post by described filter.Content should dropwise pass through described filter.

2. microcapsule bubble concentrates centrifugal scheme

2.1. under 20 ℃ (16 ℃-24 ℃) with the centrifugal 7ml/150K MWCO of 2000 * g Pierce post 60 minutes or until volume is down to 250-300 μ l.If necessary, with other 15 minutes increments, carry out centrifugal to reach volume required.

2.2., when centrifugal end, mix 15 * (avoiding producing bubble) with pipettor and take out volume (300 μ L or still less) and be transferred in new 1.7mL copolymerization property management on described post.

2.3. the final volume of described plasma extraction thing depends on the original volume of blood plasma.If described primitive plasma volume is 1ml, by plasma extraction to 300 μ l.If the primitive plasma volume is lower than 1ml, the enriched material volume should be consistent with this ratio.For example, if initial volume is 900 μ l, the volume of enriched material is 270 μ l.The equation of following is: x=(y/1000) * 300, wherein x is that final volume and the y of enriched material are initial blood plasma volumes.

2.4. record described sample volume and add 1 * PBS to prepare the final sample volume to described sample.

2.5. at the concentrated microcapsule bubble sample of 4 ℃ (2 ℃ to 8 ℃) lower storage.

Calculate:

1. the final volume of concentrated plasma sample

X=(y/1000) * 300, wherein x is that final volume and the y of enriched material are initial blood plasma volumes.

embodiment 8: use multiple method to determine the biological marking of prostate cancer

The sample that method described in use embodiment 3 is obtained is for the multiple analysis described in embodiment 4 and 5.Detection antibody used is CD63, CD9, CD81, B7H3 and EpCam.Capture antibody used be CD9, PSCA, TNFR, CD632 *, B7H3, MFG-E8, EpCam2 *, CD63, Rab, CD81, SETAP, PCSA, PSMA, 5T4, Rab IgG (contrast) and IgG (contrast), thereby obtain 100 kinds of combinations to be screened (Figure 63 C).

Screen 10 patients with prostate cancer and 12 normal control patients.By shown in catch and/or detect the results are shown in Figure 68 and Figure 70 A of antibody.Figure 70 B shows the result of using PCSA capture antibody (Figure 70 B, left figure) or EpCam capture antibody (Figure 70 B, right figure), and uses one or more to detect the detection of antibody.Sensitivity and the specificity of various combination are shown in Figure 73.

embodiment 9: use multiple method to determine the biological marking of colorectal carcinoma

The sample that method described in use embodiment 1-3 is obtained is for the multiple analysis described in embodiment 4 and 5.Detection antibody used is CD63, CD9, CD81, B7H3 and EpCam.Capture antibody used be CD9, PSCA, TNFR, CD632 *, B7H3, MFG-E8, EpCam2 *, CD63, Rab, CD81, SETAP, PCSA, PSMA, 5T4, Rab IgG (contrast) and IgG (contrast), thereby obtain 100 kinds of combinations to be screened (Figure 64 B).

Screen 10 patients with prostate cancer and 12 normal control patients.Acquired results is shown in Figure 68 and Figure 70 A.Figure 70 B shows the result of using PCSA capture antibody (Figure 70 B, left figure) or EpCam capture antibody (Figure 70 B, right figure), and uses one or more to detect the detection of antibody.Sensitivity and the specificity of various combination are shown in Figure 73.

embodiment 10: use the magnetic capture vesica

Use is according to the vesica of embodiment 2 described separation.The described vesica of about 40ul and Dynal pearl (Invitrogen, Carlsbad, CA) and the 50 μ l parent material pieces (Starting Block) of approximately 5ug (～50 μ l) EpCam antibody coating are hatched together.Under 45 ℃, described vesica and pearl are carried out to 2 hours oscillation incubations in the oscillation incubation device.The pipe that the Dynal pearl is housed is placed 1 minute on magnetic separator, and removed supernatant liquor.By described pearl washing 2 times, and all remove supernatant liquor at every turn.By pearl washing 2 times, and abandoning supernatant all at every turn.

embodiment 11: to the detection of mRNA transcript in vesica

Use Qiagen miRneasy ^tMtest kit (numbering No.217061), separate the RNA in conjunction with vesica from the pearl of embodiment 10 according to the specification sheets of manufacturers.

At QIAzol ^tMthe described vesica of homogenate in lytic reagent (Qiagen numbers No.79306).After adding chloroform, by centrifugal, even compound is separated into to water and organic phase.RNA is allocated in upper aqueous phase, and DNA in the middle of being allocated in mutually in and protein partitioning in bottom organic phase or centre mutually in.Upper aqueous phase is extracted, thereby and add ethanol to provide suitable conjugation condition for all RNA molecules that surpass 18 Nucleotide (nt).Then, described sample is applied to RNeasy ^tMon the Mini centrifugal column, wherein whole RNA is attached on film, and effectively washes away phenol and other pollutent.Then, the high-quality RNA of wash-out in not containing the water of RNA enzyme.

Use the Taqman TMPRSS:ERG syzygy transcript analytical method (Neoplasia.2007 such as Kirsten D.Mertz March; 9 (3): the RNA that 200-206.) measures the vesica of catching from the VCAP pearl.Use Taqman SPINK1 transcript analytical method (the Cancer Cell such as Scott A.Tomlins June 13 (6) in 2008: the RNA that 519-528) measures the vesica of catching from the 22Rv1 pearl.In addition, measure GAPDH transcript (control transcripts) for two groups of vesica RNA.

Higher CT value shows lower transcript expression.The variation of 1 unit of cycle threshold (CT) is equal to 2 multiple variation, and the CT difference of 3 units is equal to multiple variation of 4 etc., and it can calculate by following formula: 2^ ^cT1-CT2..The equivalent (Figure 75) that this experiment demonstrates the CT difference of syzygy transcript TMPRSS:ERG expression and uses IgG2 negative control pearl to catch.In the 22RV1 vesica, the identical of SPINK1 transcript relatively demonstrates, and changes 70.5 6.14 CT difference (Figure 75 C) for multiple.It is similar using the result of GAPDH.

embodiment 12: from the experimenter, obtain serum sample

Blood is collected in the Vacutainer SST plus blood collection tube (BD367985 or BD366643, BD Biosciences) of EDTA pipe, citrate tube or 10ml from experimenter (health volunteer and the experimenter who suffers from cancer).In latter 2 hours of collection, processing blood is to carry out separating plasma.

Sample is at room temperature placed at least 30 minutes, the longest 2 hours.By under 4 ℃ with 1,000-1, the centrifugal 15-20 of 300 * g minute and complete the separation to blood clot.Remove serum deprivation and it is scattered in 500-750 μ l freeze pipe with 500 μ l equal portions.-80 ℃ of lower stored samples.

At given setting (sitting), the blood flow volume of extraction can be between～20 to～90ml.To converge from the blood of several EDTA pipes and be transferred to without in the 50-ml of RNA enzyme/DNA enzyme cone bottom tube (Greiner), and in Hettich Rotanta 460R desktop type whizzer at room temperature with 1,200 * g centrifugal 10 minutes.Blood plasma is transferred in new pipe, stays the blood plasma supernatant liquor of level altitude of 0.5cm to avoid the disturbance agglomerate above described agglomerate.Divide sample by the blood plasma equal portions, put upside down mixing between each equal portions, and be stored in-80 ℃.

embodiment 13:RNA separates from human plasma and serum sample

Melt the human plasma of 400 μ l or serum on ice and use isopyknic 2 * denaturing soln (Ambion) to its cracking.Use liquid sample scheme (Ambion) isolation of RNA of mirVana PARIS test kit according to manufacturers, thereby described scheme is through revising with twice, isopyknic acid-phenol chloroform (as the Ambion test kit provides) extraction sample.Use the Ambion elute soln eluted rna of 105 μ l according to the scheme of manufacturers.The average-volume of the elutriant reclaimed from each post is about 80 μ l.

Also used the amplified version of described mirVana PARIS (Ambion) scheme: the blood plasma that melts 10ml on ice, the aliquots containig of 2 parts of 5-ml is transferred in the pipe of 50-ml, use isopyknic mirVana PARIS 2 * denaturing soln (Ambion) to be diluted, within 30 seconds, fully mix by vortex, and hatch on ice 5 minutes.Subsequently acid/the phenol/chloroform of equal-volume (10ml) is added in each aliquots containig.Resulting solution carries out the vortex of 1 minute and in the JA17 rotor under 20 ℃ with 8,000rpm centrifugal 5 minutes.Repeating described acid/phenol/chloroform extracts 3 times.The water volume produced fully mixes and flows through successively mirVana PARIS post with 700-μ l aliquots containig with the pure level of 100% molecule of 1.25 times of volumes ethanol.Wash described post and with elution buffer (95 ℃) eluted rna of 105 μ l according to the scheme of manufacturers.Use Nanodrop to carry out quantitatively the elutriant that amounts to 1.5 μ l.

embodiment 14: use qRT-PCR to miRNA water in the RNA from blood plasma and serum flat measurement

The fixed volume 1.67 μ l RNA solution of the pact of separating from the RNA of given sample～80 μ l elutriants as the input thing for reverse transcription (RT) reaction.For the sample from 400-μ l blood plasma or serum sample isolation of RNA, for example, 1.67 μ l RNA solution have represented the RNA corresponding to (1.67/80) * 400=8.3 μ l blood plasma or serum.For the typical curve of the RNA oligonucleotide that generates the chemosynthesis corresponding with known miRNA, thereby prepared different dilution each oligonucleotide in water, make to enter the volume that final input thing that RT reacts has 1.67 μ l.Input thing RNA is used TaqMan miRNA reverse transcription test kit and miRNA specificity stem ring primer (Applied BioSystems) reverse transcription in small-scale RT reaction, and described reaction is by 1.387 μ l H ₂o, 0.5 μ l10 * reverse transcription damping fluid, 0.063 μ l RNA enzyme inhibitors (20 units/μ l), 0.05 μ l 100mM dNTP (containing dTTP), 0.33 μ l Multiscribe reversed transcriptive enzyme and 1.67 μ l input thing RNA form; Composition except described input thing RNA can prepare the main mixture that becomes comparatively large vol, and reaction is used Tetrad2 Peltier thermal cycler (BioRad) to carry out lower 5 minutes of 16 ℃ lower 30 minutes, 42 ℃ lower 30 minutes and 85 ℃.PCR in real time is carried out on Applied BioSystems 7900HT thermal cycler, 95 ℃ lower 10 minutes, succeeded by 95 ℃ of 40 circulations lower 15 seconds lower 1 minute with 60 ℃.Use SDS relative quantification software, version 2 .2.2 (Applied BioSystems) analytical data, it uses automatic Ct to be provided for the threshold value of specifying baseline and Ct to measure.

Also can revise described scheme to comprise pre-amplification step, such as for detection of miRNA.The aliquots containig of the undiluted RT product of 1.25-μ l is mixed to produce the 5.0-μ l PCR that increases in advance with the pre-amplification PCR reagent [the TaqMan PreAmp master mixture (2 *) that every secondary response comprises 2.5 μ l and 0.2 * TaqMan miRNA Assay (being diluted in TE) of 1.25 μ l] of 3.75 μ l, its Tetrad2 Peltier thermal cycler (RioRad) upper by be heated to 95 ℃ 10 minutes, succeeded by lower 4 minutes of 95 ℃ of 14 circulations lower 15 seconds and 60 ℃, carry out.Dilute described pre-amplification PCR product (by adding the H of 20 μ l in the pre-amplification reaction product to described 5 μ l ₂o), subsequently the described dilution of 2.25 μ l is introduced in described PCR in real time and according to described and carried out.

embodiment 15: for the generation of the typical curve of miRNA absolute quantitation

Corresponding to the synthesizing single-stranded RNA oligonucleotide of ripe miRNA sequence (miRBase Release v.10.1) purchased from Sigma.To synthesize miRNA input RT and react to generate the typical curve that each miRNA TaqMan for above enumerating analyzes in the copy number scope of gained rule of thumb.Each accurate quantification lower limit of analyzing produces the Ct value in the typical curve linearity range based on input RT reaction usually and described Ct value also is not equal to or specify higher than the minimum number of copies of inputting the Ct that thing obtains by the RT of low copy number more.The Ct numerical value of use within linearity range is to the data fitting straight line from each dilution series, and equation y=mln (the x)+b that wherein derives carries out quantitatively with the absolute miRNA copy (x) for to from each sample Ct (y).According to the material in the described RT of input reaction corresponding to the known knowledge of the RNA of the total initial volume of blood plasma from 2.1% [, 1.67 total RNA effluent volume of μ l (average 80 μ l) is transfused in the RT reaction], the absolute copy number of miRNA of inputting described RT reaction is converted into the miRNA copy number of every microlitre blood plasma (or serum).The example of synthetic miRNA sequence is for miR-141, and its commercially available acquisition, such as deriving from Sigma (St.Louis, MO).

embodiment 16: from vesica, extract microRNA

According to described herein, microRNA extracts from the vesica of patient's sample from separating.For example,, referring to embodiment 7,49.Provided herein for separating of the method with concentrating vesicles.Method in the present embodiment also can be used for from patient's sample separating microRNA and without separating in advance vesica.

use the scheme of Trizol

This programme will be from Qiagen Inc., and the QIAzol lytic reagent of Valencia CA and RNeasy Midi test kit are for extracting microRNA from concentrated vesica.The step of described method comprises:

1. to the RNaseA that adds 2 μ l in 50 μ l vesica enriched materials, under 37 ℃, hatch 20 minutes.

2. the QIAzol lytic reagent that adds 700 μ l, vortex 1 minute.Add after QIAzol the Caenorhabditis elegans microRNA of 25fmol/ μ L (1 μ l) is mixed in sample, for each gross sample prepares the filler (adding up to three equal portions) of 75fmol/ μ L.

3. under 55 ℃, hatch 5 minutes.

4. add 140 μ l chloroforms and thermal agitation 15 seconds.

5. at cooled on ice 2-3 minute.

6. under 4 ℃ with 12,000 * g centrifugal 15 minutes.

7. water (300 μ L) be transferred in new pipe and add the 100%EtOH (that is, 450 μ L) of 1.5 times of volumes.

8. maximum 4ml samples are sucked to the RNeasy Midi centrifugal column (will merge from the lysate of 3 part of 50 μ l enriched material) that is placed in the 15ml collection tube.

9. at room temperature with 2700 * g centrifugal 5 minutes.

10. discard percolation liquid (flowthrough) from described centrifugal.

11. add in post by the RWT damping fluid of 1ml and at room temperature with 2700 * g centrifugal 5 minutes.The RW1 damping fluid provided in the Midi test kit is not provided.The RW1 damping fluid can wash away miRNA.The RWT damping fluid provides in the Mini test kit from Qiagen Inc.

12. discard percolation liquid.

13. add on described post by the RPE damping fluid of 1ml and at room temperature with 2700 * g centrifugal 2 minutes.

14. repeating

step

12 and 13.

16. insert in new 15ml collection tube by post and add the 150ul elution buffer.At room temperature hatch 3 minutes.

17. at room temperature with 2700 * g centrifugal 3 minutes.

18. vortex sample and being transferred in the 1.7mL pipe.By the sample storage extracted in-80 ℃.

use the scheme of MagMax

This programme will be from Applied Biosystems/Ambion, Austin, the MagMAX of TX ^tMthe RNA separating kit extracts microRNA for the vesica from concentrated.The step of described method comprises:

1. the QIAzol lytic reagent that adds 700ml, and vortex 1 minute.

2. at room temperature on experiment table, hatch 5 minutes.

3. add 140 μ l chloroforms and thermal agitation 15 seconds.

4. hatch 2-3 minute on experiment table.

5. under 4 ℃ with 12,000 * g centrifugal 15 minutes.

6. be transferred in deep-well plates by water and add 100% Virahol of 1.25 times of volumes.

7. MagMAX vibrates ^tMin conjunction with the hole of bead.The RNA of 10 μ l is sucked to each hole in conjunction with pearl.

8. collect two wash-out plates and two other deep-well plates.

9. a wash-out plate is designated as to " Elution " and another is designated as to " Tip Comb ".

10. a deep-well plates be designated as to " 1st Wash 2 " and another be designated as to " 2nd Wash 2 ".

11. fill the Wash 2 of 150 μ l in two Wash 2 deep-well plates, guarantee to add ethanol to be washed in advance.In the hole identical with sample size, filled.

12. select suitable collection procedure on MagMax Particle Processor.

13. press startup and load each suitable plate.

14. sample is transferred to micro-centrifuge tube.

15. vortex and be stored in-80 ℃.Pearl that should be residual as seen in sample.

embodiment 17: the microRNA array

Can use the microRNA level in array format (comprising high-density and low density array) analytic sample.Array analysis is used under required setting and finds differential expression, for example, by analyze the expression of multiple miR in two samples and carry out statistical study with determine those described sample room differential expression and can be therefrom for the miR of the biological marking.Described array also can be used for identifying existence or the level of one or more microRNAs in simple sample, in order to be tested and appraised the biological marking in described sample, characterizes phenotype.The present embodiment has been described the system of commercially available acquisition, and it can be used for implementing method of the present invention.

taqman low density array

As required TaqMan low density array (TLDA) miRNA card is used for to the relatively expression of each different sample sets miRNA.Use is from Applied Biosystems, Foster City, the TaqMan of CA microRNA is analyzed and the described miRNA of array system Collection and analysis.The Megaplex provided according to manufacturers ^tMpools Quick Reference Card scheme is used the TaqMan of Applied Biosystems

people's microRNA array.

exiqon mIRCURY LNA microRNA

As required by Exiqon miRCURY LNA ^tMuniversal RT microRNA PCR Human Panels I and II (Exiqon, Inc, Wobum, MA) are for the relatively miRNA expression of each different sample sets.Described Exiqon 384 hole flat boards comprise 750 kinds of miR.Sample is carried out to normalization method for the contrast primer, and described contrast primer is for the synthetic RNA filler (spike-in) from Universal cDNA synthetic agent box (UniSp6 CP).Result is carried out to normalization method for calibrate probe between plate.

To arbitrary system, implemented quality control standard (quality control standards).Three data sets of normalized value by each probe on to(for) each index is in addition average.The probe had higher than 20% average CV% is not used in analysis.Result is carried out to paired t-test to find the miR of differential expression between two sample sets.Use Benjamini and the check of Hochberg false discovery rate to proofread and correct the P value.Use GeneSpring software (Agilent Technologies, Inc., Santa Clara, CA) analytical results.

embodiment 18: the microRNA spectrum in vesica

Described according to embodiment 1-3, collect vesica by ultracentrifugation by 22Rv1, LNCaP, Vcap and normal plasma (by 16 donors, converge and obtain).Use Exiqon miR separating kit (coding No.300110,300111) to extract RNA.Analyze and measure the vesica (30 μ g) that uses equivalent according to BCA.

Reverse transcription reaction by equal-volume (5 μ l) for microRNA.By this reverse transcriptase reaction thing 81 μ l containing not diluting in the water of nuclease, then to each, independent miR adds this solution of 9 μ l in analyzing.It is found that MiR-629 only expresses in PCa (prostate cancer) vesica, and in fact can not detect in normal blood plasma vesica.It is found that MiR-9 crosses expression (measure according to copy number, exceed normally～704 times) at all PCa clone camber, and there is extremely low expression in normal blood plasma vesica.The miRNA that front 10 species diversity are expressed is shown in Figure 76.

embodiment 19: the microRNA spectrum of the vesica that magnetic EpCam catches

The pearl of embodiment 10 is placed in to QIAzol in conjunction with vesica ^tMin Lysis Reagent (Qiagen numbering 79306).125fmol Caenorhabditis elegans (c.elegans) miR-39 that adds equal portions.Use Qiagen miRneasy ^tMtest kit (numbering 217061), according to the explanation isolation of RNA of manufacturers, and at 30ul not containing wash-out in the water of RNA enzyme.

Use Veriti 96 hole thermal cyclers, the RNA of 10 μ l purifying is placed in the pre-amplification reactant of miR-9, miR-141 and miR-629.Use the pre-expansion solubilising liquid of dilution in 1: 5 to set up the qRT-PCR reaction for miR9 (ABI 4373285), miR-141 (ABI 4373137) and miR-629 (ABI 4380969) and Caenorhabditis elegans miR-39 (ABI 4373455).The result of the relative Caenorhabditis elegans of result of each sample is carried out to normalization method.

the microRNA spectrum of the vesica that embodiment 20:CD9 catches

The pearl that the EpCam used in Dynal pearl (Invitrogen, Carlsbad, the CA) alternate embodiment 19 of using CD9 to apply applies.Together with the pearl that the vesica that derives from vesica, LNCaP or the normal purifying of patients with prostate cancer is applied with CD9, hatch, and according to the described isolation of RNA of embodiment 19.Detect the expression of miR-21 and miR-141 by qRT-PCR, and acquired results is depicted in Figure 77 and Figure 78.

embodiment 21: use the vesica that filtration module carries out to separate

6mL PBS is added in 1mL blood plasma.By 1.2 microns (μ m) Pall syringe filters, described sample directly is placed in to 100kDa MWCO (Millipore, Billerica, MA), has the 7ml post (Pierce of 150kDa MWCO subsequently rockford, IL), there is the 15ml post (Millipore, Billerica, MA) of 100kDa MWCO or there is the 20ml post (Pierce of 150kDa MWCO rockford, IL) in.

Pipe carries out centrifugal 60 to 90 minutes until volume is about 250 μ l.Collect retentate and add PBC described sample is adjusted to the highest 300 μ l.Subsequently the sample of 50ul is used for to further vesica analysis, such as the analysis be further described in following examples.

embodiment 22: the multiple analysis that the vesica separated using filter carries out

Described according to embodiment 21, will use described method obtains vesica sample for multiple analysis herein.For example,, referring to embodiment 31-33.Described capture antibody is CD9, CD63, CD81, PSMA, PCSA, B7H3 and EpCam.According to Figure 79,80 and 81 described, described detection antibody is for biomarker CD9, CD81 and CD63 or B7H3 and EpCam.

embodiment 23: the vesica of the patient's sample that uses filter to carry out separates

Method by embodiment 21 is used the 7mL Pierce with 150kDa MWCO (numbering 89920/89922) the vesica sample that thickener obtains is used to multiple analysis as herein described.For example,, referring to embodiment 31-33.Described capture antibody is CD9, CD63, CD81, PSMA, PCSA, B7H3 and EpCam.Described detection antibody is CD63, CD9 and CD81.Described the results are shown in Figure 82.

embodiment 24: use filter to be separated the ratio between the vesica separated with the use ultracentrifugation ?

The vesica sample that the 500 μ l posts that use has a 100kDa MWCO obtain with the described method of embodiment 21 is used to multiple analysis as herein described.For example,, referring to embodiment 31-33.Described capture antibody is CD9, CD63, CD81, PSMA, PCSA, B7H3 and EpCam.Described detection antibody is CD63, CD9 and CD81.Acquired results is shown in Figure 83 and Figure 84.Each figure has shown and has used the different analytical procedures of implementing from single patient's sample.In two accompanying drawings, described figure has been described A) sample of ultracentrifugation purifying; B) sample filtered; C) ultracentrifugation purification of samples and 10ug Vcap and D) there is the sample of the filtration of 10ug Vcap.

embodiment 25: the sample filter relatively

Multiple filter can be used for removing large fragment from described plasma sample.Shown in table 18 and 19, size filter of filter between 1.2 and 0.8 microns provides suitable result.

Table 18: the filter of tested person

The dealer	Numbering	Film	Hole size
				Pall	4190		1.2uM
Millipore	SLAA033SB		0.8uM
				Millipore	SLAA033SS		0.8uM
GVS	FJ25ANCCA012CC01		1.2uM
				Whatman	6822-1312	GF/C glass fibre (13mm)	1.2uM
Whatman	6750-2510	Nylon	1.0uM
				Whatman	6781-2510	PES	1.0uM
Whatman	10462261	Cellulose acetate (30mm)	1.2uM
				Whatman	6783-2510	The GD glass fibre	1.0uM

According to shown in table 19, filter plasma sample and use biomarker CD9, PSMA, PCSA, CD63, CD81, B7H3 and EpCam to detect vesica.Method is according to described herein.For example,, referring to embodiment 31-33.Sample is carried out in duplicate.The result of described various filters is suitable in each mark.

Table 19: use various filters to separate the MFI of vesica

	CD9	PSMA	PCSA	CD63	CD81	B7-H3	EpCam
								High	28303	23417	22815	28630	28513	24045	27389
Blank	23	10	9	216	7	8	18

Pall?4190	726	15	152	1562	2250	172	94
								Pall?4190	787	18	173	1701	2539	208	100
GVS	631	17	163	1722	2738	182	86
								GVS	562	19	164	1504	2315	206	95
SLAA033SB	739	26	243	1521	2078	277	135
								SLAA033SB	725	23	160	1316	1790	263	116
SLAA033SS	888	19	191	1384	2025	223	124
								SLAA033SS	774	22	173	1392	2063	218	100
6822-1312	824	19	196	1601	2359	207	106
								6822-1312	697	18	172	1632	2580	202	93
10462261	576	19	178	1495	2420	190	93
								10462261	553	23	191	1465	2091	221	106
6750-2510	743	32	248	1574	2082	286	144
								6750-2510	847	38	250	1545	2019	322	171
6783-2510	853	19	178	1474	2185	219	110
								6783-2510	757	20	159	1533	2232	199	103
6781-2510	624	23	202	1433	2189	227	111
								6781-2510	711	21	196	1365	1966	218	131

embodiment 26: to the flow cytometry of vesica

Use MoFlo XDP (Beckman Coulter, Fort Collins, CO, USA) to analyze the blood plasma vesica of purifying and used Summit 4.3 softwares (Beckman Coulter) to analyze the meta fluorescence intensity.Use the direct mark vesica of antibody, maybe can add pearl or microballoon (as, the magnetic polystyrene during 7 looks that comprise BD FACS arrange, number 335775).Can use the wedding agent for following vesica antigen to detect vesica: CD9 (mouse anti human CD9, MAB1880, R& D Systems, Minneapolis, MN; USA), PSM (mouse anti human PSM, sc-73651, Santa Cruz; Santa Cruz, CA, USA), PCSA (mouse anti human prostatic cell surface antigen; MAB4089, Millipore, MA; USA), CD63 (mouse anti human CD63,556019, BD Biosciences; San Jose; CA, USA), CD81 (mouse anti human CD81,555675; BD Biosciences; San Jose, CA, USA), B7-H3 (goat anti human B 7-H 3; AF1027, R& D Systems, Minneapolis, MN, USA), EpCAM (mouse anti human EpCAM, MAB9601, R& D Systems, Minneapolis, MN, USA).Can use the fluorescent-labeled antibody for required vesica antigen to detect vesica: for example, FITC, phycoerythrin (PE) and Cy7 are generally used for the described antibody of mark.

For using multiple microballoon capture antibody, described microballoon can be available from Luminex (Austin, TX, USA) and use Sulfo-NHS and EDC available from Pierce Thermo (to be respectively numbering No.24510 and No.22981, Rockford, Ill, USA) be coupled to required antibody with micros.

At room temperature the vesica of purifying (10ug/ml) and 5,000 microballoons are carried out to 1 hour oscillation incubation.Under 1700rpm, use FACS damping fluid (0.5%FBS/PBS) to wash described sample 10 minutes.At room temperature described detection antibody is carried out to one hour oscillation incubation by manufacturers's recommended density.After another time of using the FACS damping fluid to carry out with 1700rpm 10 minutes washed, described sample is resuspended in 100ul FACS damping fluid and on the FASC instrument and moves.

In addition, when using microballoon to detect vesica, the vesica be labeled can detect antibody component according to it and be sorted in different pipes.For example, by using the microballoon of FITC or PE mark, the first pipe comprises does not have a microballoon colony of detection agent, and the second pipe comprises the colony with PE detection agent, the 3rd pipe comprises the colony with FITC detection agent, and the 4th pipe comprises the colony with PE and FITC detection agent.The vesica colony of institute's sorting can be subject to further analysis, for example, by detecting useful load (such as mRNA, microRNA or protein content), is analyzed.

Figure 85 shows and uses the vesica that MoFlo XDP carries out separate and identify.In the experiment of this group, there are approximately 3000 trigger events (microparticle that is about large vesica size) of only having a buffer reagent.There is approximately the be unstained trigger event (43,000 sizes are enough to the vesica of scattering laser) of vesica of 46,000 tools.There is the trigger event of 500,000 tool dyeing vesicas.Use has detected vesica for the detection agent (all with FITC, carrying out mark) of four transmembrane protein CD9, CD63 and CD81.Than vesicles, can when being subject to detection agent dyeing, it be detected.

Figure 86 A show use the Cy7 mark anti-psca antibody from patients with prostate cancer blood plasma to the airflow classification of vesica.By airflow classification, the per-cent of PCSA+ vesica is increased to 68% from 35%.The anti-CD45 antibody that Figure 86 B shows applying marking from normal patient and patients with prostate cancer blood plasma to the airflow classification of vesica.Compare with normal healthy controls, have the CD45+ vesica per-cent that exceeds 5 times in described cancer blood plasma.After airflow classification, the per-cent of CD45+ vesica improves a lot.Because CD45 is the immunne response mark, the immunity raising that is derived from vesica has shown the immunne response in described patients with prostate cancer.The anti-CD45 antibody that Figure 86 C shows applying marking from normal patient and plasma of breast cancer patients to the airflow classification of vesica.Compare with normal healthy controls, exist in described mammary cancer blood plasma and exceed the CD45+ vesica per-cent that surpasses 10 times.After sorting, the per-cent of CD45+ vesica improves a lot.Because CD45 is the immunne response mark, the immunity raising that is derived from vesica has shown the immunne response in described patient with breast cancer.The anti-DLL4 antibody that Figure 86 D shows applying marking from normal patient and patients with prostate cancer blood plasma to the airflow classification of vesica.Compare with normal healthy controls, in described prostate cancer blood plasma, exist and exceed the approximately DLL4+ vesica per-cent of 10 times.By airflow classification, the per-cent of DLL+ vesica improves a lot.The DLL4+ vesica improved can show the vascularization strengthened in described cancer patients.

The physical sepn of being undertaken by the sorting to specific vesica colony contributes to other research, such as to the partially or completely microRNA analysis of the vesica colony of purifying.

embodiment 27: the antibody test of vesica

Use the techniques described herein, the vesica detected in patient's sample by the pearl with the antibody coating is assessed the vesica in described sample.Used following overall plan:

A. care location (as, clinical clinic, doctor working space, hospital) extract blood from the patient.

B. the blood plasma of described blood is partly for further analysis.

C. for to remove macrobead and to separate the part that comprises vesica, plasma sample is filtered to (syringe filter that for example, uses 0.8 or 1.2 micron (μ m)) also subsequently by size exclusion post (as having the molecular weight cutoff value of 150kDa).Overall diagram has been shown in Figure 87 A.Shown in Figure 87 B, filtering is preferred with respect to ultracentrifugation.Not bound by theory, high speed centrifugation is anchored to the protein target in film a little less than can removing, this is contrary with four transmembrane proteins that are anchored to more securely in film, and high speed centrifugation can reduce the cell-specific target in vesica, and it will not be detected in the subsequent analysis of the biological marking to described vesica.

D. described vesica part and the pearl be coupled to for " catching " antibody of target marker thing are hatched always.Use subsequently " detection " antibody (as the antibody of phycoerythrin or FITC coupling) through mark to tag to the vesica of catching.Described pearl also can carry out mark.

E. detect in described sample and catch and tagged vesica.Can be according to detecting fluorescently-labeled pearl shown in Figure 78 C and detecting antibody.The use of the detection antibody of the pearl of mark and mark allows to be assessed having with the pearl of the vesica of its combination by capture antibody.

F. to data analysis.Can be for meta fluorescence intensity (MFI) setting threshold of specific capture antibody.This capture antibody can mean specific phenotype higher than the reading of described threshold value.As illustrative example, for the capture antibody of cancer markers, higher than the MFI of threshold value, can mean the existence of cancer in patient's sample.

In Figure 87, described pearl 816 is flow through kapillary 811.The use of the dual laser apparatus 812 of different wave length allows to detect independently at detector 813 places capture antibody 818 (by being derived from the fluorescent signal of described pearl) and meta fluorescence intensity (MFI) (the detection antibody 819 by mark produces).As directed, from the use of the pearl (each pearl carries out mark with different fluorescence) of the mark of different target capture antibody coupling, allow, in single analysis, different vesica 817 colonies are carried out to multiple analysis.Laser 1815 allows pearl type (that is, capture antibody) is detected, and laser 2814 allows to be measured detecting antibody, and it can comprise general vesica mark, such as four transmembrane proteins (comprising CD9, CD63 and CD81).The use of different pearl colonies and laser allows, in single analysis, multiple different vesica colony is carried out to multiple analysis simultaneously.

embodiment 28: the vesica reference point of prostate cancer

14 3 phase prostate cancer experimenters, 11 benign prostatic hyperplasia (BPH) samples and 15 normal specimens are tested.Use the method described in embodiment 3 to obtain the vesica sample, and for multiple analysis, described in embodiment 4 and 5.Sample analyzed determined to 4 standards: 1) whether described sample had the vesica of expressing; 2) whether described sample had the prostate gland vesica of expressing; 3) whether described sample had the cancer vesica of expressing; And 4) whether described sample is reliable.If sample meets 4 all standards, sample is classified as to the prostate cancer positive and there is different sensitivity and specificity, it depends on according to the biological marking of the existing difference of the shown sample of table 20.

In this table, " vesica " listed threshold value or the reference point of vesica level, " prostate gland " listed for the threshold value of prostate gland vesica or reference point, threshold value or the reference point of 3 kinds of biological markings of difference of prostate cancer listed in " cancer-1 ", " cancer-2 " and " cancer-3 ", threshold value or reference point or the reliability of qualitative contrast listed on " QC-1 " and " QC-2 " hurdle, and specificity (" Spec ") and sensitivity (" Sens ") for benign prostatic hyperplasia (BPH) have been listed in 4 last hurdles.

Table 20: sensitivity and the specificity of the biological marking of prostate cancer

As follows for four kinds of standards that described sample is classified:

Vesica is crossed expression

Sample meta fluorescence intensity (MFI) in three kinds of analyses is for measuring the value of described sample.Each is analyzed and has used different capture antibodies.The CD9 capture antibody has been used in the first analysis, and the CD81 capture antibody has been used in the second analysis, and the third analyzes use CD63 antibody.Identical detection antibody combination (for the antibody of CD9, CD81 and CD63) is analyzed for each.If described three kinds of mean values of analyzing gained are greater than 3000, this sample is classified as and had the vesica (table 20, vesica) of expressing.

Crossing of prostate gland vesica expressed

Sample MFI in two analyses is averaged to measure to the value of described sample.Each is analyzed and has used different capture antibodies.The first has been used the PCSA capture antibody, and the second has been used the PSMA capture antibody.The like combinations of the detection antibody of mark (for the antibody of CD9, CD81 and CD63) is analyzed for each.If these the two kinds mean values of analyzing gained are greater than 100, this sample is classified as and had the prostate gland vesica (table 20, prostate gland) of expressing.

Crossing of cancer vesica expressed

Determine with 3 kinds of biological markings of different cancers in sample, whether the cancer vesica crosses expression.The first (cancer-1) has been used the EpCam capture antibody and for the detection antibody of CD81, CD9 and CD63.The second (cancer-2) has been used the CD9 capture antibody and for the detection antibody of EpCam and B7H3.If for any two kinds of biological markings of the biological marking of three kinds of cancers, sample MFI value is higher than reference point, and this sample is classified as and had the cancer (table 20, cancer-1, cancer-2 and cancer-3) of expressing.

The reliability of sample

For each sample, measure two quality control survey values, QC-1 and QC-2.If described sample meets one of them, to be classified as be reliable to this sample.

For QC-1, measure the summation of all MFI that analyze for 7 times.The detection antibody for CD59 and PSMA is all used in each time in analyzing for 7 times.Capture antibody for each analysis is CD63, CD81, PCSA, PSMA, STEAP, B7H3 and EpCam.If this summation is greater than 4000, this sample be insecure and be not included in.

For QC-2, measure the summation of all MFI that analyze for 5 times.The detection antibody for CD9, CD81 and CD63 is all used in each time in analyzing for 5 times.Capture antibody for each analysis is PCSA, PSMA, STEAP, B7H3 and EpCam.If this summation is greater than 8000, this sample be insecure and be not included in.

After sample meets described standard, the sensitivity and the specificity that have the sample of BPH and do not have the sample of BPH are shown in Table 20.

embodiment 29: the detection of prostate cancer

High quality training collection sample is available from commercial supplier.Described sample comprises the blood plasma from 42 routine normal prostatic, 42 routine PCa and 15 routine BPH patients.Described PCa sample comprises 4 routine III phases and remaining II phase.Described sample before having worked in all experiments were chamber in blind method.

By filtering the particle that surpasses 1.5 microns to remove, then use hollow fiber film tube to carry out the centrifugal and purifying of post, thereby obtain vesica from described sample.Use the described sample of multiple analysis systems analysis based on pearl as described above.

Analyzed the antibody for following protein:

A. general vesica (MV) mark: CD9, CD81 and CD63

B. prostate gland MV mark: PCSA

C. relevant MV mark: the EpCam of cancer and B7H3

Sample is required by following quality test: if multiple meta fluorescence intensity (MFI) PSCA+MFI B7H3+MFI is EpCam<and 200, sample is discarded higher than the signal of background owing to lacking.In described training set, 6 samples (3 normal specimens and 3 prostate cancer samples) do not reach enough quality scores and are excluded.The upper limit of MFI has also been carried out following setting: if the MFI of EpCam>6300, test exceeds the scoring of this upper limit and sample and is considered as non-cancer " feminine gender " of described test purpose (that is, for).

According to 6 kinds of appraisal result of the MFI for the antibody of training set protein are classified to described sample, wherein must meet the following conditions so that sample classifies as the PCa positive:

A. the average MFI of general MV mark>1500

b.PCSA?MFI＞300

c.B7H3?MFI＞550

D.EpCam MFI is between 550 and 6300

Use described 84 normal and PCa training data samples, described test is found to have 98% sensitivity and 95% specificity for the relative normal specimens of PCA.Referring to Figure 88 A.The MFI improved than PCa sample with normal phase has been shown in Figure 88 B.Figure 89 A with sensitivity and the specificity of comparing described test with traditional PSA with PCA3 is shown respectively in 89B.With traditional PSA and PCA3 test, compare, the PCa test in the present embodiment can make 220 male sex avoid the hardship of unnecessary tissue biopsy in the normal male of every 1000 examinations.

embodiment 30: distinguish BPH and PCa

BPH is the common cause that the PSA level rises.Whether PSA only can indicate prostate gland to go wrong, but it can not distinguish BPH and PCa effectively.PCA3 it is found that it is the transcript that prostate cancer cell is crossed expression, and it is considered to have slightly high specificity for PCa, but this depends on for the threshold value of PSA and PCA3 and the colony of studying.

Can pass through vesica (MV) analysis and characterize BPH.By checking the sample described in embodiment 29,10 (67%) in 15 BPH samples have the CD63+ vesica level higher than PCa sample (comprising the III phase).Referring to Figure 90.In addition, 14 (93%) in 15 BPH have the CD63+ vesica level higher than normal specimens.The inflammation specificity marking that this prompting is different from cancer can be used for distinguishing BPH and PCa.

Repeat the test as the PCa in embodiment 29, comprise 15 BPH samples.In the situation that use whole 99 samples, described test has 98% sensitivity and 84% specificity.Referring to Figure 91.Therefore, described test provides 15% improvement with respect to PSA.The performance number of PSA and PCA3 do not reported for not having in its cohort under the setting of BPH usually, and however, vesica test of the present invention is in the situation that even comprise that BPH still is better than traditional test.Referring to Figure 92.In this arranges, with PSA test, compare, PCa test of the present invention every 1000 do not suffer from PCa screened the male sex in make 110 male sex avoid unnecessary tissue biopsy.Because obtaining positive findings, described analysis accepts in the male sex of tissue biopsy, most of prostate glands have some problem, because described test performance good (that is, thering is 95% specificity in this colony, referring to embodiment 29) aspect the evaluation normal male.

Figure 93 has provided the ROC tracing analysis of vesica analysis contrast traditional test of the present invention.When described ROC curve is soaring rapidly to the upper left corner of this figure, True Positive Rate is high and false positive rate (1-specificity) is low.AUC shown in Figure 93 relatively shows, with traditional PSA or PCA3 test, compares, and test of the present invention more may correctly be classified to sample.

Figure 94 shows, at general vesica (MV) level, prostate specific MV with have between the MV level of cancer markers and have dependency, this shows that these marks are relevant in described population of subjects.These cancer specific marks can be further used for distinguishing BPH and PCa.In the figure, general MV mark comprises CD9, CD63 and CD81; Prostate gland MV mark comprises PCSA and PSMA; And cancer MV mark comprises EpCam and B7H3.Do not use vesica to catch mark and the test of PCa sample has been represented to sensitivity and the specificity values almost identical with the test of using general MV mark to carry out.Similarly, do not use B7H3 and the cancer detection of carrying out has only caused small reduction on performance.These data show that mark of the present invention can be replaced and test under various configuration, and still realize the optimized analysis performance.

Figure 95 shows the other mark that can distinguish PCa and normal specimens, and it can add to improve test performance.The figure shows and use ICAM1, EGFR, STEAP1 and PSCA to be caught and use meta fluorescence intensity (MFI) level of carrying out the vesica of mark for the phycoerythrin traget antibody of four transmembrane protein CD9, CD63 and CD81.。

embodiment 31: microballoon vesica prostate cancer analytical plan

In the present embodiment, vesica PCa test is the immunoassay based on microballoon for detection of protein biomarker collection, and described protein biomarker is present on the vesica from the blood plasma of suffering from patients with prostate cancer.The specific antibody for following protein biomarker is used in described test: CD9, CD59, CD63, CD81, PSMA, PCSA, B7H3 and EpCAM.After the microballoon applied by antibody is caught vesica, by the antibody of phycoerythrin mark for detection of vesica specific biological mark.Referring to Figure 96 A.According to these antibody with from the vesica of patient's blood plasma in conjunction with level, carry out whether prostate cancer is existed definite.

According to the described separation vesica of embodiment 7.

microballoon

By specific antibodies and the coupling of microballoon (Luminex) phase, afterwards described microballoon is combined to prepare microballoon master mixture, it is comprised of L100-C105-01, L100-C115-01, L100-C119-01, L100-C120-01, L100-C122-01, L100-C124-01, L100-C135-01 and L100-C175-01.XMAP

classification Calibration Microspheres L100-CAL1 (Luminex) is used for Luminex LX200 instrument as equipment Alignment reagent.XMAP

reporter Calibration Microspheres L100-CAL2 (Luminex) is used for Luminex LX200 instrument as the device report calibrating reagent.XMAP

classification Control Microspheres L100-CON1 (Luminex) is used for Luminex LX200 instrument as device control reagent.XMAP

reporter Control Microspheres L100-CON2 (Luminex) controls reagent for Luminex LX200 instrument as report.

capture antibody

In the present embodiment, following antibody is caught to specific vesica colony for applying the Luminex microballoon with its corresponding protein target by conjunction with on vesica: a. mouse anti human CD9 monoclonal antibody is IgG2b, and it is for applying microballoon L100-C105 with preparation * EPCLMACD9-C105; B. mouse anti human PSMA monoclonal antibody is IgG1, and it is for applying microballoon L100-C115 with preparation EPCLMAPSMA-C115; C. mouse anti human PCSA monoclonal antibody is IgG1, and it is for applying microballoon L100-C119 with preparation EPCLMAPCSA-C119; D. mouse anti human CD63 monoclonal antibody is IgG1, and it is for applying microballoon L100-C120 with preparation EPCLMACD63-C120; E. mouse anti human CD81 monoclonal antibody is IgG1, and it is for applying microballoon L100-C124 with preparation EPCLMACD81-C124; F. the goat anti human B 7-H 3 monoclonal antibody is the IgG antibody purification, and it is for applying microballoon L100-C125 with preparation EPCLGAB7-H3-C125; And g. mouse anti human EpCAM monoclonal antibody is the IgG2b antibody purification, it is for applying microballoon L100-C175 with preparation EPCLMAEpCAM-C 175.

detect antibody

Following phycoerythrin (PE) traget antibody is used as detection probes: a.EPCLMACD81PE in this analysis: mouse anti human CD81PE traget antibody is IgG1 antibody, and it is for detection of the CD81 caught on vesica; B.EPCLMACD9PE: mouse anti human CD9PE traget antibody is IgG1 antibody, and it is for detection of the CD9 caught on vesica; C.EPCLMACD63PE: mouse anti human CD63PE traget antibody is IgG1 antibody, and it is for detection of the CD63 caught on vesica; D.EPCLMAEpCAMPE: mouse anti human EpCAM PE traget antibody is IgG1 antibody, and it is for detection of the EpCAM caught on vesica; E.EPCLMAPSMAPE: mouse anti human PSMA PE traget antibody is IgG1 antibody, and it is for detection of the PSMA caught on vesica; F.EPCLMACD59PE: mouse anti human CD59PE traget antibody is IgG1 antibody, and it is for detection of the CD59 caught on vesica; And g.EPCLMAB7-H3PE: mouse anti human B7-H3PE traget antibody is IgG1 antibody, and it is for detection of the B7-H3 caught on vesica.

the reagent preparation

antibody purification:following antibody in table 21 is carried out purifying and is adjusted to required working concentration from retailer and according to following scheme.

Table 21: the antibody of analyzing for PCa

Antibody	Purposes
		EPCLMACD9	The microballoon of catching for vesica applies
EPCLMACD63	The microballoon of catching for vesica applies
		EPCLMACD81	The microballoon of catching for vesica applies
EPCLMAPSMA	The microballoon of catching for vesica applies
		EPCLGAB7-H3	The microballoon of catching for vesica applies
EPCLMAEpCAM	The microballoon of catching for vesica applies
		EPCLMAPCSA	The microballoon of catching for vesica applies
EPCLMACD81PE	The PE detected for the vesica biomarker applies antibody
		EPCLMACD9PE	The PE detected for the vesica biomarker applies antibody
EPCLMACD63PE	The PE detected for the vesica biomarker applies antibody

EPCLMAEpCAMPE	The PE detected for the vesica biomarker applies antibody
		EPCLMAPSMAPE	The PE detected for the vesica biomarker applies antibody
EPCLMACD59PE	The PE detected for the vesica biomarker applies antibody
		EPCLMAB7-H3PE	The PE detected for the vesica biomarker applies antibody

Antibody purification scheme: use protein G resin (Protein G spin kit, the production number 89979) antibody purification from Pierce.Miniature chromatographic column that will be prepared by the P-200 suction nozzle filtered is for purifying.

Use is loaded on the protein G resin of 100 μ l in each micro-column from 100 μ l damping fluids of described Pierce test kit.Waiting several minutes so that, after described resin settled, use when needed P-200 pipettor (Pipettman) to apply air pressure to drain damping fluid, and guarantee that this post can not be dried.(pH 7.4,100mM phosphate buffered saline buffer, 150mM NaCl for the binding buffer liquid of use 0.6ml; (Pierce, production number 89979)) this post of balance.Antibody is applied to this post (antibody of<1mg is loaded on this post).Use 1.5ml binding buffer liquid to wash this post.Prepared 5 pipes (1.5ml micro-centrifuge tube) and 10 μ l neutralization solutions (Pierce, production number 89979) have been applied to each pipe.Use from the elution buffer of described test kit by antibody elution to each pipe of described 5 pipes, every pipe 100ul (amounting to 500 μ l).Use Nanodrop (Thermo scientific, Nanodrop 1000 spectrophotometers) to measure the relative absorbancy of each fraction under 280nm.Selection has the fraction of the highest OD reading and uses for downstream.Use Pierce Slide-A-Lyzer Dialysis Cassette (Pierce, production number 66333,3KDa cutoff) with 0.25 liter of PBS damping fluid described sample of dialysing.Under 4 ℃ under continuous stirring state, every 2 hours exchange buffering liquid, minimumly carry out three times and change.Subsequently dialysed sample is transferred in the 1.5ml micro-centrifuge tube, and can is identified and be stored under 4 ℃ (short-terms) or-20 ℃ (for a long time).

the assembling of microballoon workable mixtures:microballoon workable mixtures MWM101 comprises antibody, microballoon and the coating microballoon of front four rows in table 22.

Table 22: antibody-microballoon combination

Antibody	Microballoon	Apply microballoon
			EPCLMACD9	L100-C105	EPCLMACD9-C105
EPCLMACD63	L100-C120	EPCLMACD63-C120
			EPCLMACD81	L100-C124	EPCLMACD81-C124
EPCLMAPSMA	L100-C115	EPCLMAPSMA-C115
			EPCLGAB7-H3	L100-C125	EPCLGAB7-H3-C125
bEPCLMAEpCAM	L100-C175	EPCLMAEpCAM-C175
			EPCLMAPCSA	L100-C119	EPCLMAPCSA-C119

According to following scheme use above listed its separately corresponding antibody apply microballoon.

the scheme that is used for two step carbodiimide couplings of protein and carboxylation microballoon:should be protected in order to avoid be exposed to for a long time under light at microballoon described in this process.According to the coupling raw material microballoon not of the indication resuspension described in the product information page provided with described microballoon (xMAP technologies, MicroPlex TM Microspheres).By 5 * 10 ⁶individual raw material microballoon is transferred in USA Scientific 1.5ml micro-centrifuge tube.By at room temperature with >=8000 * g, carrying out the micro-centrifugal raw material microballoon that precipitates of 1-2 minute.Remove supernatant liquor and approximately 20 seconds the microballoon of precipitation was resuspended to the dH of 100 μ l by vortex and supersound process ₂in O.By at room temperature with >=8000 * g, carrying out the micro-centrifugal microballoon that precipitates of 1-2 minute.Remove supernatant liquor and approximately the microballoon through cleaning is resuspended to the 100mM SODIUM PHOSPHATE, MONOBASIC of 80 μ l 20 seconds by vortex and supersound process (Branson 1510, Branson ULTrasonics Corp.), in pH 6.2.The 50mg/ml Sulfo-NHS of 10 μ l (Thermo Scientific, number 24500) (is diluted in to dH ₂in O) add to described microballoon and gently mix by vortex.The 50mg/ml EDC of 10 μ l (Thermo Scientific, numbering 25952-53-8) (is diluted in to dH ₂in O) add to described microballoon and gently mix by vortex.At room temperature hatch described microballoon 20 minutes and gently mix by vortex with the interval of 10 minutes.By at room temperature with >=8000 * g, carrying out the micro-centrifugal microballoon activated that precipitates of 1-2 minute.Remove supernatant liquor and approximately 20 seconds described microballoon was resuspended to the 50mM MES of 250 μ l by vortex and supersound process, in pH 5.0 (MES, Sigma, numbering M2933).(only ((Sigma (P3688-10PAK+0.05% sodiumazide (S8032))) should be used as analysis buffer and lavation buffer solution PBS-1%BSA+ trinitride (PBS-BN).) precipitate described microballoon by room temperature with >=8000 * g, carrying out 1-2 minute micro-centrifugal subsequently.

Remove supernatant liquor and approximately 20 seconds described microballoon was resuspended to the 50mM MES of 250 μ l by vortex and supersound process, in pH 5.0 (MES, Sigma, numbering M2933).(only ((Sigma (P3688-10PAK+0.05% sodiumazide (S8032))) should be used as analysis buffer and lavation buffer solution PBS-1%BSA+ trinitride (PBS-BN).) precipitate described microballoon by room temperature with >=8000 * g, carrying out 1-2 minute micro-centrifugal subsequently, complete therefrom 50mM MES, twice washing that pH 5.0 carries out.

The microballoon of removing supernatant liquor and approximately will activating and wash 20 seconds by vortex and supersound process is resuspended to the 50mM MES of 100 μ l, in pH 5.0.The protein of 125,25,5 or 1 μ g amount is added to the microballoon of resuspension.(annotate: can carry out titration to measure the optimum protein quality of each concrete linked reaction in 1 to 125 μ g scope.) using 50mM MES, pH 5.0 is adjusted to 500 μ l by cumulative volume.At room temperature mix by vortex mixed linked reaction thing and by it under (by Labquake rotator, being rotated on Barnstead) and hatch 2 hours.By at room temperature with >=8000 * g, carrying out the micro-centrifugal microballoon that precipitates coupling of 1-2 minute.Remove supernatant liquor and approximately 20 seconds the microballoon of described precipitation be resuspended in the PBS-TBN of 500 μ l by vortex mixed and supersound process.(can optimize concentration for the concrete reagent used, analysis condition, multiplexing level etc.)

the microballoon analytical plan:detect the preparation of antibody according to the multiple phycoerythrin of the use of describing in embodiment 4.Above according to system handbook (high PMT arranges), analyze 100 μ l at Luminex analyser (Luminex 200, xMAP technologies).

decision tree:decision tree (Figure 96 B-D) for assessment of the result from described microballoon analysis to determine whether the experimenter suffers from cancer.Set up the threshold of MFI and according to the result of the MFI score of described antibody to sample classification, thereby determine sample whether have enough signals for implementing to analyze (as, be effective sample or be invalid sample for further analysis for analysis, can obtain in this case second patient's sample) and described sample whether be the PCa positive.Figure 96 B shows the decision tree of using the MFI obtained with CD59, PSMA, PCSA, B7-H3, EpCAM, CD9, CD81 and CD63.Figure 96 C shows the decision tree of using the MFI obtained with PSMA, B7-H3, EpCAM, CD9, CD81 and CD63.If within the standard deviation of described MFI in predetermined threshold value, sample is classified as uncertain.For being verified, when using described single four transmembrane proteins to catch vesica and use whole four transmembrane proteins to carry out mark, sample must have enough signals.If described prostate specific mark (PSMA) is considered the allied signal of positive and described cancer markers (B7-H3 and EpCam) and also is regarded as the positive, the sample that has passed through checking is called the positive.Figure 96 D shows the decision tree of using the MFI obtained with PCSA, PSMA, B7-H3, CD9, CD81 and CD63.If within the standard deviation of described MFI in predetermined threshold value (TH), sample is classified as uncertain.In this case, can obtain second patient's sample.For being verified, when using single four transmembrane proteins to catch vesica and use whole four transmembrane proteins to carry out mark, described sample must have enough signals.Be considered positive and described cancer markers (B7-H3) and also be considered in positive situation if arbitrary in described prostate specific mark (PSMA or PCSA), the sample that has passed through checking is called the positive.

result:referring to

embodiment

32 and 33.

embodiment 32: microballoon vesica PCa analytical performance

In the present embodiment, described vesica PCa test is the immunoassay based on microballoon for detection of a histone matter biomarker, and described protein biomarker is present on the vesica from the patient's who suffers from prostate cancer blood plasma.Test class is similar to the test of embodiment 31 carries out, its change shown in having hereinafter.

Described test is used and is designed for the multiple immunoassay that detects the circulation microcapsule bubble.Described test use PCSA, PSMA and B7H3 with catch the microcapsule bubble be present in patient's sample (such as blood plasma) and use CD9, CD81 and CD63 to detect the microcapsule bubble of being caught.The result of this analysis is the meta fluorescence intensity (MFI) produced by the antibody capture to microcapsule bubble (described microcapsule bubble comprises the single capture protein on this microcapsule bubble and detects protein) and fluorescently-labeled antibody test.If contain the definite threshold value of MFI horizontal exceeding experience of the microcapsule bubble of PSMA or PCSA and B7H3 albumen, according to this specimen, be " positive ".If these two kinds of microcapsule bubbles are caught any one in classification and shown the MFI level lower than the experience definite threshold, sample is defined as " feminine gender ".Perhaps, if going out excessive statistical discrepancy, the Data Representation that does not meet specific threshold value or replicate(determination) due to the MFI value make sample MFI can not produce clearly the positive or negative result, the result of report " indefinite ".Explanation for this test " can not assess " means that this patient's sample comprises for the inadequate microcapsule bubble amount of analysis.The method of the threshold value obtained for the mensuration experience is referring to embodiment 33.

The specific antibody for following protein biomarker is used in described test: as the CD9 in embodiment 31, CD59, CD63, CD81, PSMA, PCSA and B7H3.Summarize according to table 23, set decision rules for definite sample, whether to be called the positive, feminine gender or indefinite.Also referring to embodiment 31.For being called as positive sample, the test repeated must surpass whole four MFI cutoffs of determining for four transmembrane protein marks (CD9, CD63, CD81), prostate gland mark (PSMA or PCSA) and B7H3.If from two kinds in three repeated tests of PSMA and PCSA or crossed over the MFI cutoff from any one in three repeated tests of B7H3 antibody, sample is called as indefinite.If at least one in four transmembrane protein marks (CD9, CD63 and CD81), prostate gland mark (PSMA or PCSA), B7H3 be lower than the MFI cutoff, sample is called as negative.

Table 23: the MFI parameter of each capture antibody

Described vesica PCa test is compared with the PSA of raising of cohort that confirms by tissue biopsy to suffer from or do not suffer from 296 patients of Pca.The ROC curve of described result has been shown in Figure 97 A.According to demonstration, the area under curve (AUC) of vesica PCa test is 0.94, and the AUC of the PSA improved on same sample is only 0.68.The PCa sample is probably because high PSA value is found.Therefore this colony is subject to tending to twisting of PSA, and this has caused the AUC higher than true clinical setting.

Further in the group of 933 patient's plasma samples, carried out vesica PCa test.Result is illustrated in Figure 97 B and is summarized in table 24.

Table 24: the performance of vesica PCa test on 933 patient's cohort

True positives	409
		True negative	307
False positive	50
		False negative	72
Can not assess	63
		Indefinite	32
Amount to	933

Sensitivity	85％
		Specificity	86％
Accuracy	85％
		Can not assessment ratio	8％
Indefinite rate	5％

Shown in table 24, vesica PCa test has realized 85% level of sensitivity with 86% specificity level, has reached 85% accuracy.In contrast, PSA has approximately 55% specificity under 85% sensitivity, and PSA has approximately 5% sensitivity under 86% specificity.Referring to Figure 97 A.In 933 routine samples approximately 12% for can not assess or indefinite.Can heavily collect and reappraise from described patient's sample.Figure 97 C shows the ROC curve corresponding to the data shown in Figure 97 B.Vesica PCa test has 0.92 AUC for described 933 routine samples.

embodiment 33: meta fluorescence intensity (MFI) threshold calculations

Usually set the threshold level of biomarker, wherein higher or lower than the value representation otherness result of described threshold value, as the positive to negative findings.For example, the threshold value that the standard of PSA is 4ng/ml PSA in serum.PSA level lower than this threshold value is considered to normally, and can indicate prostatic problem higher than the PSA value of this threshold value, for example BPH or prostate cancer (PCa).Adjustable thresholds is beneficial to strengthen sensitivity with respect to specificity.In the situation of PSA, lower threshold test goes out more cancer, and has therefore improved sensitivity, but can increase false-positive quantity simultaneously and reduce thus specificity.Similarly, higher threshold test goes out less cancer, and has therefore reduced sensitivity, but can reduce false-positive quantity simultaneously and improve thus specificity.

In the embodiment of this paper (such as embodiment 29-32), for vesica biomarker setting threshold MFI value to build the test for detection of PCa.This embodiment provides the approach of determining described threshold value.For this purpose, use cluster analysis to determine whether to exist the PCa positive and negative colony, it can be separated according to the MFI threshold value that produces required level of sensitivity.

The fluorescence intensity level exponentially distributes, and therefore before carrying out described cluster analysis, data is carried out number conversion.Resulting data set carries out traditional definite clustering method (hard clustering method).Definite cluster that carry out in this place is used the Euclidean distance parameter (Euclidean distance parameter) of definition whether to belong to specific cluster with specified data point.The algorithm used each data point is distributed to one of c cluster so that in cluster sum of squares minimize:

Σ_{i = 1}^{c} \underset{k &Element; A_{i}}{Σ} {| | x_{k} {- v}_{i} | |}_{2}

A wherein _ithe collection of object in the i cluster (data point), and v _ithe mean value of upper those points of cluster i.This equation represents the Euclidean distance norm.Data from 149 routine samples are used for hard clustering.Raw data is carried out number conversion so that it is uniformly distributed.Subsequently by deduct minimum value and divided by maximum value by data normalization.PSMA mapping to PCSA to B7H3 and PSMA to B7H3, PCSA before conversion has been shown in Figure 98 A and after conversion.

Various may the combination of mark (PSMA to B7H3, PCSA to B7H3 and PSMA to PCSA) analyzed, and definite threshold carries out optimal separation with the group to being confirmed.Wherein find two kinds of clusters have been carried out the horizontal and vertical lines of optimal separation.The crossing point of this line and axle is used for defining cutoff, and at first this need described value is gone to normalization method, gets subsequently antilogarithm.This has produced respectively 90 and 300 cutoff to B7H3 for each PSAM, shown in Figure 98 B.

For PCSA, to B7H3, two kinds of clusters finding are shown in Figure 98 C.Wherein find described two kinds of clusters are carried out the horizontal and vertical lines of optimal separation.The crossing point of this line and axle is used for defining cutoff, and at first this need described value is gone to normalization method, gets subsequently antilogarithm.This has produced respectively 430 and 300 cutoff to B7H3 for each PCSA.

For PSMA, to PCSA, two kinds of clusters finding are shown in Figure 98 D.Wherein find described two kinds of clusters are carried out the horizontal and vertical lines of optimal separation.The crossing point of this line and axle is used for defining cutoff, and at first this need described value is gone to normalization method, gets subsequently antilogarithm.This has produced respectively 85 and 350 cutoff to PCSA for each PSMA.

The sensitivity of all threshold value combination calculation and the specificity for described cluster analysis, found.For 85 or 90 the value of PSMA, sensitivity and specificity are unchanged, and therefore 90 is selected with being cutoff.For 430 or 350 the threshold value of PCSA, sensitivity is unchanged, although specificity reduces by 0.3% with changing.Because this is non-noticeable change, selected 350 value for the PCSA cutoff, thereby made error in the higher sensitivity side.Two kinds of clusters have identical threshold value 300 for B7H3, therefore use this value.The sensitivity and the specificity that with these threshold values, obtain are respectively 92.7% and 81.8%.

These threshold application are in the more large data sets that comprises 313 routine samples, and have produced 92.8% sensitivity and 78.7% specificity.Referring to Figure 98 E.

Whether the threshold value in the present embodiment is to determine from normal person or the irrelevant mode of cancer patients with described sample.Because the performance in separating two colonies of described threshold value is good, therefore probably due to the biology difference of sample, in fact there are two independently basic populations.This difference to heavens with the existence of prostate cancer height correlation whether, and therefore as the good guide of carrying out tissue biopsy.Described method is determined the MFI threshold value for the comparison for any hope, such as other cancer outside detecting normally.

embodiment 34: the discovery of vesica PCa protein marker

In the present embodiment, assess as described above vesicle protein matter biomarker.Sample, from amounting to 522 patients, comprises 285 routine prostate cancer samples and 237 example contrasts.The mark of testing comprises CD9, PSMA, PCSA, CD63, CD81, B7H3, IL 6, OPG-13, IL6R, PA2G4, EZH2, RUNX2, SERPINB3 and EpCam.Result is shown in Figure 99, and it shows average fluorescent strength (MFI) value of prostate cancer and normal specimens for each test mark.Observed the vesica surface marker of higher level for the whole marks four transmembrane proteins (as CD63 and CD81) except as general vesica biomarker.The performance of various marks and combination thereof is shown in table 25.

Table 25: the performance of various marks and mark combination

Mark	Sensitivity	Specificity
			PSMA
	84％	81％
			PCSA	87％	82％
B7-H3	81％	85％
			IL?6	83％	82％
OPG-13	91％	81％
			IL6R
	84％	81％
			PA2G4	90％	80％

EZH2	81％	89％
			RUNX2
	94％	74％
			SERPINB3	96％	68％
OPG+PA2G4+RUNX2+SERPI	93％	81％
			PCSA+B7H3	81％	87％
PA2G4+RUNX2+SERPI	88％	82％
			OPG+RUNX2+SERPI	88％	85％
OPG+PA2G4+SERPI	86％	85％
			OPG+PA2G4+RUNX2	85％	86％
?OPG+PA2G4+RUNX2+SERPI+PCSA+B7H3	94％	79％

As the analysis of the various marks in the present embodiment and combination thereof for finding to detect the vesica mark of disease.Except analytical performance, the selection of antibody can be subject to the perceptibility of described mark in medical field, reagent operability, carry out the ability of multiple analysis and the impact of other factors.

embodiment 35: for detection of the vesicle protein array of prostate cancer

In the present embodiment, implement vesica PCa test by using protein array (more specifically, antibody array) detection to be present in from protein biomarker collection on the vesica of the blood plasma of suffering from patients with prostate cancer.Described array comprises the specific capture antibody of following protein biomarker: CD9, CD59, CD63, CD81.According to separation vesica mentioned above, as, in embodiment 3 and 7.Vesica at the male sex's to from there being the PCa risk (such as the male sex who surpasses 50 years old) blood plasma is filtered and after separating, and plasma sample is hatched together with the array that carries various capture antibodies.That according to the fluorescent mark for PSMA, PCSA, B7H3 and EpCAM, detects antibody carries out whether determine of prostate cancer existence in conjunction with level, and described antibody combines in the vesica of described array with the hybridization from patient's blood plasma.

In the second array format, vesica from blood plasma, separate and with the hybridization array that comprises CD9, CD59, CD63, CD81, PSMA, PCSA, B7H3 and EpCAM.Use tags to the vesica of catching with the non-specific vesica antibody of Cy3 and/or Cy5 mark.Detect fluorescence.The pattern that depends on combination, carry out whether determine of prostate cancer existence.

embodiment 36: distinguish BPH and PCa on antibody array

With dilution in 1: 30, comprise from the concentrated blood plasma of the vesica of 8 BPH and 8 III phase patients with prostate cancer and by itself and the Raybiotech Human Receptor hybridization array that comprises 40 kinds of human cell factor acceptors.Use non-paired t test to compare the mean concns (pg/ml) for each cytokine of each group.In 40 kinds of acceptors of test, Trappin-2, Ceacam-1, HVEM, IL-10Rb, IL-1R4 and BCMA be differential expression the most significantly.Referring to Figure 100.

Statistical significance by the t check for definite differential expression.Result is illustrated in table 26.

Table 26: the BPH that uses the antibody mark to carry out and the differentiation of Pca

Mark	The p value
		Trappin-2	0.018
Ceacam-1	0.013
		HVEM	0.0095
IL-10Rb	0.052
		IL-1R4	0.054
BCMA	0.019

Distinguish BPH and PCa by the combination of using 6 kinds of marks, for BPH, with 87.5% specificity, obtained 100% sensitivity.

embodiment 37: use miR to distinguish BPH and PCa

Analyze the RNA of the source plasma vesica of and 9 individualities of suffering from 3 phase prostate cancers individual from 9 normal males on Exiqon mIRCURY LNA microRNA PCR system group.Exiqon 384 orifice plate groups are measured 750 kinds of miR.Sample is carried out to normalization method with respect to the contrast primer of the synthetic RNA filler (spike-in) for Universal cDNA synthetic agent box.Normalized value by each probe on three data sets of each indication (indication) (BPH or PCa) is in addition average.The probe had higher than 20% average CV% is not used in analysis.

The analysis of described result has been disclosed and compared in the BPH sample 2 times or cross higher the multiple microRNA of expressing with 3 phase prostate cancer samples.These miR comprise: hsa-miR-329, hsa-miR-30a, hsa-miR-335, hsa-miR-152, hsa-miR-151-5p, hsa-miR-200a and hsa-miR-145 as shown in Table 27ly go out:

Table 27: the miR that crosses expression in BPH with respect to PCa

Cross expression with respect to PCa in BPH	Multiple changes
		hsa-miR-329	12.32
hsa-miR-30a	6.16
		hsa-miR-335	6
hsa-miR-152	4.73
		hsa-miR-151-5p	3.16

hsa-miR-200a	3.16
		hsa-miR-145	2.35

In addition, a large amount of miR crosses and expresses with respect at least 2 times of ground of BPH in Pca.These miR comprise: hsa-miR-29a, hsa-miR-106b, hsa-miR-595, hsa-miR-142-5p, hsa-miR-99a, hsa-miR-20b, hsa-miR-373, hsa-miR-502-5p, hsa-miR-29b, hsa-miR-142-3p, hsa-miR-663, hsa-miR-423-5p, hsa-miR-15a, hsa-miR-888, hsa-miR-361-3p, hsa-miR-365, hsa-miR-10b, hsa-miR-199a-3p, hsa-miR-181a, hsa-miR-19a, hsa-miR-125b, hsa-miR-760, hsa-miR-7a, hsa-miR-671-5p, hsa-miR-7c, hsa-miR-1979 and hsa-miR-103, as shown in Table 28ly go out:

Table 28: cross the miR expressed in PCa vs BPH

Cross expression with respect to BPH in PCa	Multiple changes
		hsa-miR-29a	2.18
hsa-miR-106b	2.23
		hsa-miR-595	2.24
hsa-miR-142-5p	2.25
		hsa-miR-99a	2.3
hsa-miR-20b	2.36
		hsa-miR-373 ^*	2.37
hsa-miR-502-5p	2.39
		hsa-miR-29b	2.43
hsa-miR-142-3p	2.44
		hsa-miR-663	2.51
hsa-miR-423-5p	2.55
		hsa-miR-15a	2.71
hsa-miR-888	2.72
		hsa-miR-361-3p	2.86
hsa-miR-365	2.9
		hsa-miR-10b	2.9
hsa-miR-199a-3p	2.96
		hsa-miR-181a	3
hsa-miR-19a	3.03
		hsa-miR-125b	3.05
hsa-miR-760	3.1
		hsa-miR-7a	3.77
hsa-miR-671-5p	4.11

hsa-miR-7c	5.56
		hsa-miR-1979	5.8
hsa-miR-103	6.42

embodiment 38: the miR-145 in contrast and PCa sample

Figure 101 shows the comparison of miR-145 in contrast and prostate cancer sample.Collect as described in example 13 above RNA.Contrast comprise PSA<4ng/ml and the optimum digital rectal examination>Caucasian of 75 years old and>the non-descendants American of 65 years old.As seen in Fig., miR-145 expresses not enough in the PCa sample.MiR-145 can be used for suffering from respect to the Individual identification with benign prostate variation (as BPH) individuality of in early days/latent (indolent) Pca.

embodiment 39: the discovery of the miR of differential expression in transitivity and non-metastatic PCa

The panel of 720 kinds of miR is for relatively expressing from 48 patients of the non-metastatic prostate cancer of suffering from confirmation and 19 patients' the miR of plasma sample that suffers from the metastatic prostate cancer of confirmation.Be derived from the RNA of the microcapsule bubble of described plasma sample in 1.0 editions (Exiqon microRNA ready to use qRT-PCR panel version 1.0) upper evaluations of Exiqon microRNA instant qRT-PCR group.Result is also used it for paired t-test subsequently with respect to calibrate probe normalization method between plate.Use Benjamini and the check of Hochberg false discovery rate to proofread and correct the p value.

Table 29 shows front four kinds of raise most and the front four kinds of miR that lower most that identify like this.In other target mRNA, miR-145 is predicted regulation and control BRAF (its oncogene that is well-characterized) also.MiR-32 and miR-134 prediction regulation and control SMAD6 (its negative regulator to BMP and TGF-β/activator signal transduction is relevant).SMAD6 expresses associated with bad cancer prognosis.

Table 29: transitivity PCa sample is expressed with respect to the miR of non-metastatic PCa sample

Use Exiqon microRNA instant qRT-PCR group 2.0 editions (Exiqon microRNA ready to use qRT-PCR panel version 2.0) and repeat above-mentioned experiment setting from 10 patients that suffer from metastatic prostate cancer with the plasma sample of 17 patients' that suffer from the non-metastatic prostate cancer cohort.The non-correction p value of the miR expressed on significant difference ground has been shown in table 30.

Table 30: the miR with respect to non-metastatic PCa sample in transitivity PCa sample expresses

embodiment 40: the detection of the miR of differential expression in PCa

MiR panel is for relatively expressing from 28 male sex that do not suffer from prostate cancer and the miR of plasma sample that suffers from 64 male sex of prostate cancer.In all cases, confirm the prostate cancer state by tissue biopsy.Estimate the RNA of the microcapsule bubble that is derived from described plasma sample on Exiqon microRNA instant qRT-PCR group.Paired t-test is also used it in result calibrate probe normalization method between plate subsequently.Use Benjamini and the check of Hochberg false discovery rate to proofread and correct the p value.The p value of the miR of differential expression the most significantly has been shown in table 31.

Table 31: the miR with respect to non-metastatic PCa sample in transitivity PCa sample expresses

MicroRNA	The p value	Adjusting in contrast	Multiple changes
				hsa-miR-574-3p	0.0264	Lower	3.52
hsa-miR-331-3p	0.0264	Lower	6.63

hsa-miR-326	0.0264	Lower	5.99
				hsa-miR-181a-2 ^*	0.0264	Raise	2.95
hsa-miR-130b	0.0264	Lower	4.83
				hsa-miR-301a	0.0264	Lower	8.18
hsa-miR-141	0.0312	Lower	4.13
				hsa-miR-432	0.0351	Lower	3.83
hsa-miR-107	0.0351	Lower	8.29
				hsa-miR-628-5p	0.0391	Raise	3.25
hsa-miR-625 ^*	0.0391	Lower	3.98
				hsa-miR-497	0.0495	Lower	4.35
hsa-miR-484	0.0495	Lower	2.9

embodiment 41: the miR of differential expression in PCa

According to use described herein Exiqon RT-PCR group analysis the panel of 84 routine prostate cancers and 28 routine control samples (tissue biopsy's contrast of not suffering from prostate cancer).Use the TNM scoring, prostate cancer comprises 13 routine MX samples (not estimating far away the transfer), 55 routine M0 samples (without far shifting) and 16 routine M1 samples (transfer far away of confirmation).

Detect miR in separation in the vesica of described patient's sample.Use is through improved Qiagen miRneasy scheme (Qiagen GmbH, Germany) isolation of RNA from the 150 μ l frozen plasma enriched materials from the various kinds product.Thereby process described concentrated sample with RNaseA before described improved plan is included in and separates is only analyzed the RNA be protected in vesica in each sample.Sample is mixed to Caenorhabditis elegans microRNA with known quantity with the normalization method for subsequent step.The 40ng RNA that uses the vesica from sample to separate to each Exiqon group.

Described Exiqon RT-PCR group is comprised of two 384 cards, and it has contained 750 kinds of miR and check analysis.Analyze and implement described qRT-PCR analysis at the upper Sybr of use of ABI 7900 (Life Technologies Corporation, Carlsbad, California) green.Between the relative plate of Ct value that each miR is analyzed, normalization method is carried out in the Ct value of calibration (IPC) probe and RT-PCR contrast.Several quality tests are inserted.Remove sample when IPC Ct value>25, RT-PCR Cr value>35 and when sample does not increase contrast miR (being miR-16 and miR-21) from analyze.Use GeneSpring software (Agilent Technologies, Inc., Santa Clara, CA) to carry out the principle component analysis of sample data to identify outlier.Owing to failing up to standard with these quality metrics and removed three samples from described analysis.

According to hereinafter illustrating, data are carried out between sample sets to paired t-test and with Benjamini; The p value is proofreaied and correct in the check of Hochberg false discovery rate.Use Taqman probe method proof list to reveal the miR of the most remarkable p value.

84 routine prostate cancers and 28 example contrasts are compared.On the level between described PCa and control sample, 81 (81) in 750 kinds of miR probes planted have>2.0 multiple and changed (lowering and 75 kinds of rises for 6 kinds).In these 81 kinds, 10 kinds of have<correction p values of 0.05.Referring to table 32.

MiR with respect to control sample in table 32:PCa sample expresses

MicroRNA	The p value	Adjusting in the PCa sample	Multiple changes
				hsa-miR-574-3p	0.0339	Raise	3.38
hsa-miR-141	0.0339	Raise	4.26
				hsa-miR-331-3p	0.0442	Raise	5.53
hsa-miR-432	0.0442	Raise	3.32
				hsa-miR-326	0.0339	Raise	5.1
hsa-miR-2110	0.0339	Raise	6.71
				hsa-miR-107	0.0317	Raise	11.31
hsa-miR-130b	0.0339	Raise	4.66
				hsa-miR-301a	0.0442	Raise	5.21
hsa-miR-625 ^*	0.0442	Raise	3.55

In the situation that containing 16 routine M1 transitivity samples, do not repeat comparison as above.This has relatively determined in 750 kinds of miR 81 kinds of miR of have on the level between described PCa and control sample>2.0 multiples variations.Referring to table 33." adjusting in contrast " refers in control sample with respect to the rise (rising) of PCa sample or lowers (decline).

MiR with respect to control sample in table 33:PCa sample (non-metastatic sample) expresses

MicroRNA	Multiple changes	Adjusting in contrast
			hsa-miR-107	12.78	Lower
hsa-miR-2110	6.42	Lower
			hsa-miR-326	5.75	Lower
hsa-miR-301a	4.87	Lower
			hsa-miR-185	4.41	Lower
hsa-miR-331-3p	4.28	Lower
			hsa-miR-373 ^*	4.11	Lower
hsa-miR-99a	4.06	Lower
			hsa-miR-432	3.96	Lower
hsa-miR-625 ^*	3.83	Lower

hsa-miR-130b	3.6	Lower
			hsa-miR-638	3.56	Lower
hsa-miR-425 ^*	3.55	Lower
			hsa-miR-627	3.53	Lower
hsa-miR-197	3.46	Lower
			hsa-miR-532-3p	3.45	Lower
hsa-miR-124	3.42	Lower
			hsa-miR-411 ^*	3.42	Lower
hsa-miR-154	3.4	Lower
			hsa-miR-16-2 ^*	3.24	Lower
hsa-miR-574-3p	3.23	Lower
			hsa-miR-421	3.21	Lower
hsa-miR-18a	3.17	Lower
			hsa-miR-141	3.12	Lower
hsa-miR-423-3p	3.09	Lower
			hsa-miR-103	3.05	Lower
hsa-miR-28-3p	3.01	Lower
			hsa-miR-375	3	Lower
hsa-miR-765	2.77	Lower
			hsa-miR-362-5p	2.77	Lower
hsa-miR-22 ^*	2.75	Lower
			hsa-miR-181b	2.74	Lower
hsa-miR-186	2.74	Lower
			hsa-miR-652	2.69	Lower
hsa-miR-192	2.61	Raise
			hsa-miR-518f ^*	2.61	Lower
hsa-miR-1207-5p	2.59	Lower
			hsa-miR-532-5p	2.58	Lower
hsa-miR-484	2.56	Lower
			hsa-miR-577	2.51	Raise
hsa-miR-379 ^*	2.51	Lower
			hsa-miR-363	2.51	Lower
hsa-miR-1224-3p	2.49	Lower
			hsa-miR-210	2.46	Lower
hsa-miR-181a-2 ^*	2.44	Raise
			hsa-miR-19b	2.41	Lower

hsa-miR-604	2.4	Lower
			hsa-miR-125a-5p	2.39	Raise
hsa-miR-1	2.38	Lower
			hsa-miR-518c ^*	2.36	Lower
hsa-miR-95	2.33	Lower
			hsa-miR-140-5p	2.33	Lower
hsa-miR-497	2.31	Lower
			hsa-miR-491-5p	2.28	Lower
hsa-miR-144	2.26	Lower
			hsa-miR-18b	2.25	Lower
hsa-miR-423-5p	2.25	Lower
			hsa-miR-665	2.25	Lower
hsa-miR-324-3p	2.25	Lower
			hsa-miR-335	2.24	Lower
hsa-miR-590-5p	2.2	Lower
			hsa-miR-130a	2.19	Lower
hsa-miR-133b	2.18	Lower
			hsa-miR-1972	2.16	Lower
hsa-miR-744 ^*	2.15	Lower
			hsa-miR-202	2.14	Raise
hsa-miR-30e	2.12	Lower
			hsa-miR-214	2.1	Lower
hsa-miR-29c	2.1	Lower
			hsa-miR-20a	2.1	Lower
hsa-miR-1247	2.09	Raise
			hsa-miR-15b ^*	2.08	Lower
hsa-miR-133a	2.08	Lower
			hsa-miR-194	2.04	Lower
hsa-miR-26b ^*	2.04	Lower
			hsa-miR-191	2.04	Lower
hsa-miR-106b	2.03	Lower
			hsa-miR-485-3p	2.03	Lower
hsa-miR-1909	2.02	Lower
			hsa-miR-628-5p	2.02	Raise
hsa-miR-431	2	Lower

In 81 kinds of miR of table 33,9 kinds of have<not correction p values of 0.01.In PCa, all raised.Referring to table 34." adjusting " refers in the PCa sample with respect to the rise (rising) of control sample or lowers (decline).

MiR with respect to control sample in table 34:PCa sample (non-metastatic sample) expresses

miR	The p value	Regulate	Multiple changes
				hsa-miR-107	0.0002	Raise	12.78
hsa-miR-326	0.0015	Raise	5.75
				hsa-miR-432	0.0024	Raise	3.96
hsa-miR-574-3p	0.0029	Raise	3.23
				hsa-miR-625 ^*	0.0038	Raise	3.83
hsa-miR-2110	0.0044	Raise	6.42
				hsa-miR-301a	0.0079	Raise	4.87
hsa-miR-141	0.0087	Raise	3.12
				hsa-miR-373 ^*	0.009	Raise	4.11

In the further checking to the above results, from the microcapsule bubble of the contrast male sex (without PCa) that confirm through tissue biopsy from 35 and 133 male sex's that suffer from the non-metastatic prostate cancers blood plasma extraction, extract microRNA.MicroRNA is used ABI Taqman dissecting needle to the expression evaluation of miR-107 and miR-574-3p and uses synthetic typical curve to carry out absolute quantitation to copy number.Between the RNA separation period, use the 75 Caenorhabditis elegans miR-39 that fly mole as filler, sample separates difference for RNA and carries out normalization method.Use Mann-Whitney U check to compare the result from each group.Result is illustrated in Figure 102 A (miR-107) and Figure 102 B (miR-574-3p).As each figure below indicates, the p value is significantly different between contrast and PCa sample, has verified thus the result obtained with the Exiqon card shown in table 32-34.When compare and PCa sample, also similarly in experiment, with Taqman, verifying miR-141.

Repeated comparison as above between 16 routine M1 transitivity PCa samples and 55 routine M0 non-metastatic PCa samples.This Identification 121 kinds of miR changing of have on the level between transitivity and non-metastatic sample in 750 kinds of miR>2.0 multiple.Referring to table 35." adjusting in non-metastatic " refers in non-metastatic PCa sample with respect to the rise (rising) of transitivity PCa sample or lowers (decline).

MiR with respect to M0 non-metastatic PCa in table 35:M1 transitivity PCa expresses

In 121 kinds of miR of table 35,9 kinds of have<p values of 0.01.Referring to table 36.In transitivity PCa sample, 9 kinds are rise.

MiR with respect to M0 non-metastatic PCa in table 36:M1 transitivity PCa expresses

miR	The p value	Adjusting in metastatic cancer	Multiple changes
				hsa-miR-200b	0.0007	Raise	4.75
hsa-miR-375	0.0011	Raise	12.75
				hsa-miR-582-3p	0.002	Raise	2.26
hsa-miR-17 ^*	0.0023	Raise	5.65
				hsa-miR-1296	0.0034	Raise	3.6
hsa-miR-20a ^*	0.0039	Raise	3.19
				hsa-miR-100	0.0061	Raise	3.97
hsa-miR-452	0.0091	Raise	4.17
				hsa-miR-577	0.0098	Raise	4.88

MiR-17 ^*and miR-20a ^*be arranged in oncomir1 bunch.

Taqman analyzes in transitivity Pca situation, with respect to non-metastatic PCa situation, having verified several miR.Figure 103 A-D shows in the level of separating miR-141 (Figure 103 A), miR-375 (Figure 103 B), miR-200b (Figure 103 C) and miR-574-3p (Figure 103 D) in the vesica of transitivity (M1) and non-metastatic (MO) prostate cancer sample.In all cases, the p value is significant when the level compared between transitivity and non-metastatic sample.

The summary of Taqman the result has been shown in table 37.In this table, M0 and M1 refer to for testing the sample number of various microRNAs.The p value is all significant in all cases.

Table 37: the miR with respect to M0 non-metastatic PCa in the M1 transitivity PCa obtained by Taqman expresses

miR	M0	M1	The p value
				hsa-miR-200b	73	33	0.05
hsa-miR-375	71	33	0.0001
				hsa-miR-141	73	39	0.0001
hsa-miR-331-3p	64	27	0.002
				hsa-miR-181a	65	27	0.002
hsa-miR-574-3p	65	32	0.0001

The level from the hsa-miR-141 of the RNA of serum source vesica in the independent cohort of 47 bit transition patients with prostate cancer and hsa-miR-375 also found is significantly higher than the level (p=0.0001) in 72 non-recurrence patients with prostate cancer.

Vesica from blood plasma and serum is the reliable sources for the microRNA of biomarker.With contrasting of confirming through tissue biopsy, compare, two kinds of miR (hsa-miR-107 and 574-3p) are found in the prostate cancer sample and raise especially.In metastatic source plasma vesica sample, it is found that several miR raise significantly, and find that 2 kinds (hsa-miR-141 and hsa-miR-375) in these miR raise especially in transitivity serum source vesica.

embodiment 42: for strengthening the miR of vesica diagnositc analysis performance

According to described herein, vesica concentrates and is assessed to provide the result output of diagnosis, prognosis or treatment diagnosis in patient's plasma sample.According to described herein, the vesica analysis of patient's sample comprises the detection to vesica surface biological mark (as surface antigen) and/or vesica useful load (as mRNA and microRNA).Can assess useful load in vesica to strengthen analytical performance.For example, Figure 104 A shows the miR used in vesica and analyzes false negative is converted into to the schematic diagram of the scheme of true positives, has improved thus sensitivity.In this scheme, according to the vesica surface antigen, analysis is called as the useful load in vesica and further turned out to be true negative or true positives by assessment of negative sample.Similarly, Figure 104 B shows the miR used in vesica and analyzes false positive is converted into to the schematic diagram of the scheme of true negative, has improved thus specificity.In this scheme, according to the vesica surface antigen, analysis is called as the useful load in vesica and further turned out to be true negative or true positives by assessment of positive sample.

Diagnostic test for prostate cancer comprises the vesica whether existed to detect the indication prostate cancer from the separation of the blood sample from patient vesica.For example,, referring to embodiment 27-34.Described blood can be serum or blood plasma." capture antibody " of identifying specific vesica surface antigen by use caught separates vesica.Comprise four transmembrane protein CD9, CD63 and CD81 (it usually is present on the vesica of blood and therefore plays a role as general vesica biomarker), prostate specific biomarker PSMA and PCSA and cancer specific biomarker B7H3 for the surface antigen of Diagnosis of prostate cancer.Described capture antibody mooring is on fluorescently-labeled pearl, and wherein said pearl carries out the otherness mark for each capture antibody.Use further highlights the vesica of catching for four transmembrane protein CD9, CD63 and CD81 fluorescently-labeled " detection antibody ".From the fluorescence of described pearl and described detection antibody for the vesica amount measuring plasma sample and express described surface antigen with for described Diagnosis of prostate cancer.Fluorescence level in sample and reference level (it can be different from the sample with prostate cancer) are compared.In the present embodiment, the microRNA analysis is for strengthening the performance of the Diagnosis of prostate cancer based on vesica.

Figure 104 C shows by the detected result of miR-107 in the sample of the assessment of the Diagnosis of prostate cancer based on vesica.Figure 104 D shows by the detected result of miR-141 in the sample of the assessment of the Diagnosis of prostate cancer based on vesica.In the figure, the normalization method level of the miR indicated illustrates on Y-axis, and it is the alleged false positive (FP) of the alleged true positives of vesica diagnositc analysis (TP), vesica diagnositc analysis alleged true negative (TN), vesica diagnositc analysis and the alleged false negative (FN) of vesica diagnositc analysis.Shown in Figure 104 C, the use of miR-107 has been strengthened by false negative and true negative distinguishing to (p=0.0008) sensitivity that vesica is analyzed.Similarly, Figure 104 D also shows, the use of miR-141 has been strengthened by false negative and true negative distinguishing to (p=0.0001) sensitivity that vesica is analyzed.The result of adding miR-141 has been shown in table 38.The performance of miR-574-3p is similar.

Table 38: in the test of the PCa based on vesica, add miR-141

Without miR-141	There is miR-141
		Sensitivity
85％	98％
		Specificity	86％	86％

In the present embodiment, by the surface antigen of indicating prostate cancer, detect vesica, and further support the performance of the described marking by detecting the miR in vesica, that is, in the situation that to specificity, do not cause disadvantageous effect to improve sensitivity.Can be expanded with for wherein for surface antigen or out of Memory feature, vesica being carried out to somatotype this basic skills, subsequently by one or more other biomarker for strengthening any situation of phenetic analysis.Herein, described one or more other biomarkers are miR.Any other vesica associated biomolecule entity that it also can comprise mRNA, soluble proteins, lipid, carbohydrate and can be used for characterizing the target phenotype.

embodiment 43: the comparison of miR express spectra in blood plasma, serum and clone vesica

By Agilent v3miRNA microarray (Agilent Technologies, Inc., Santa Clara, CA) for comparing the expression from the patient's who suffers from prostate cancer blood plasma and the vesica source microRNA (miR) between serum, normal healthy controls, a kind of prostate cancer cell line and tumor of prostate and healthy tissues.Use Qiagen miReasy test kit to separate total RNA from blood plasma with the clone vesica, and use Exomir extraction method (Bioo Scientific Corp., Austin, TX) to separate total RNA from serum.Each sample of 100ng and described microarray hybridization and use GeneSpring software package are analyzed to obtained data.The hierarchical cluster that sample is carried out with gene has shown unique express spectra of comparing the blood plasma vesica with clone source vesica with tumor tissues.For the microRNA of differential expression significantly and estimated the serum and plasma from patients with prostate cancer and normal control.The vesica that is derived from peripheral blood provides unique source for the miR based on blood analyzes.

embodiment 44: the separation of vesica subgroup and miR somatotype subsequently

In the present embodiment, detect microRNA (miR) express spectra in circulation microcapsule bubble subgroup, described microcapsule bubble subgroup forms and defines according to surface protein.Use method as herein described, the vesica formed separating from prostate cancer cell line (VCaP) according to its surface protein carries out airflow classification.Described vesica is for the differential expression of miR and estimated.To be used for for the phycoerythrin traget antibody of EpCam, CD63 or B7-H3 the sorting vesica subgroup by the cell sorting of fluorescence-activation.At the upper sorting vesica of Beckman-Coulter MoFlo XDP (Beckman Coulter, Inc., Brea, CA), thereby make each vesica can be used as individual particle and be analyzed.Due to the abundance of antigen on the vesica surface, there is the significant intensity skew with respect to the isotype contrast on the FL2 passage.The subgroup of sorting is expressed and somatotype according to miR subsequently.The miR somatotype of the positive subgroup of EpCam, CD6 or B7-H3 and the somatotype of total VCaP vesica colony are compared.Observe the miR express spectra of difference between subgroup, and all express spectras are from viewed different in total group.Observe the mistake of miR and express and express not enough pattern between group.The subgroup of these data presentation vesicas can be distinguished and be separated according to surface protein mark and gene element (being miR in this case) thereof.According to surface protein form from patient's blood plasma chorista specificity vesica colony and according to the ability that surface protein forms and gene element is analyzed it, can be used for subsequently in diagnosis as herein described, prognosis or treat diagnostic use.

embodiment 45: the microRNA biomarker in the male sex with prostate cancer and low PSA

Although with low PSA (as, lower than 4ng/ml) the increase prostate gland of combination may indicate benign prostatic hyperplasia (BPH), these clinical observations indication prostate cancers but not BPH in some cases.The biomarker that can distinguish these two groups will allow have low PSA the symptom male sex is arranged in realize the early detection to prostate cancer.

Use method as herein described, from 13 control patients from thering is PSA<4.0ng/ml (the 1st group), have PSA >=4.0ng/ml 15 control patients (the 2nd group), there are 9 non-metastatic patients with prostate cancer (the 3rd group) of PSA<4.0ng/ml and have the plasma sample of 59 non-metastatic patients with prostate cancer (the 4th group) of PSA >=4.0ng/ml and separate vesica.According to described herein, separate the microRNA useful load and use the Exiqon RT-PCR group formed by 750 kinds of miR probes to detect it from described vesica.Normalized result is at the patients with prostate cancer with PSA>4.0 (the 4th group) and have between the patients with prostate cancer (the 3rd group) of PSA<4.0 and compare.Between these groups, 344 kinds of miR probes are found to have the multiple that surpasses 2 times to be changed.Referring to table 39.In table 39, " multiple variation " refers to the variation of level between the 3rd and the 4th group, and " adjusting " refers to the rise (rising) of comparing with the 3rd group in the 4th group and lower (decline).

Table 39:PSA<or >=the PCa sample of 4.0ng/ml in the multiple of miR level change

MicroRNA	Multiple changes	Regulate
			hsa-miR-143	41.44	Lower
hsa-miR-432	31.23	Lower
			hsa-miR-425	18.66	Lower
hsa-miR-32	17.55	Lower
			hsa-miR-424	15.78	Lower
hsa-miR-96	14.72	Lower
			hsa-miR-629	14.54	Lower
hsa-miR-532-5p	13.43	Lower
			hsa-miR-215	13.4	Lower

hsa-miR-920	12.21	Lower
			hsa-miR-421	11.78	Lower
hsa-miR-204	11.18	Lower
			hsa-miR-29a	11.04	Lower
hsa-miR-148a	10.62	Lower
			hsa-miR-19b	10	Lower
hsa-miR-595	9.96	Lower
			hsa-miR-590-5p	9.93	Lower
hsa-miR-518f	9.76	Lower
			hsa-miR-518f ^*	9.68	Lower
hsa-miR-766	9.49	Lower
			hsa-miR-22 ^*	9.01	Lower
hsa-miR-491-5p	8.95	Lower
			hsa-miR-29b	8.83	Lower
hsa-miR-144	8.52	Lower
			hsa-miR-451	8.33	Lower
hsa-miR-376a	8.27	Lower
			hsa-miR-577	8.22	Lower
hsa-miR-151-3p	8.18	Lower
			hsa-let-7f	7.74	Lower
hsa-miR-188-3p	7.52	Raise
			hsa-let-7i	7.52	Lower
hsa-miR-19a	7.45	Lower
			hsa-miR-616 ^*	7.35	Lower
hsa-miR-140-3p	7.28	Lower
			hsa-miR-18a ^*	7.24	Lower
hsa-miR-154 ^*	7.11	Lower
			hsa-miR-423-5p	7.11	Lower
hsa-miR-192	7.01	Lower
			hsa-miR-212	7	Lower
hsa-miR-107	6.95	Lower
			hsa-miR-16-2 ^*	6.89	Lower
hsa-miR-205	6.84	Lower
			hsa-miR-199a-3p	6.84	Lower
hsa-miR-101	6.78	Lower
			hsa-miR-130a	6.75	Lower

hsa-miR-15a	6.64	Lower
			hsa-miR-363	6.58	Lower
hsa-miR-30b ^*	6.56	Lower
			hsa-miR-146b-5p	6.54	Lower
hsa-miR-142-5p	6.48	Lower
			hsa-miR-197	6.44	Lower
hsa-miR-339-5p	6.41	Lower
			hsa-miR-140-5p	6.39	Lower
hsa-miR-450a	6.27	Lower
			hsa-miR-624 ^*	6.21	Lower
hsa-miR-122	6.15	Lower
			hsa-miR-665	6.13	Lower
hsa-miR-125b	6.06	Lower
			hsa-miR-937	6.05	Lower
hsa-miR-148b	5.99	Lower
			hsa-miR-106b ^*	5.99	Lower
hsa-miR-769-5p	5.95	Lower
			hsa-miR-1255b	5.94	Lower
hsa-miR-517 ^*	5.87	Lower
			hsa-miR-517a	5.85	Lower
hsa-let-7d	5.68	Lower
			hsa-miR-365 ^*	5.67	Lower
hsa-miR-302d ^*	5.57	Lower
			hsa-miR-221 ^*	5.54	Lower
hsa-miR-103-2 ^*	5.47	Lower
			hsa-miR-136	5.43	Lower
hsa-let-7g	5.43	Lower
			hsa-miR-424 ^*	5.25	Lower
hsa-miR-124	5.12	Lower
			hsa-miR-103	5.1	Lower
hsa-miR-23b ^*	5.09	Lower
			hsa-miR-191	5.08	Lower
hsa-miR-221	5.06	Lower
			hsa-miR-324-5p	5.06	Lower
hsa-miR-330-5p	5.03	Lower
			hsa-miR-302b	5.01	Lower

hsa-miR-570	4.96	Lower
			hsa-miR-105 ^*	4.95	Lower
hsa-miR-15b	4.95	Lower
			hsa-miR-29c	4.91	Lower
hsa-miR-497	4.76	Lower
			hsa-miR-617	4.74	Lower
hsa-miR-1200	4.69	Lower
			hsa-miR-29a ^*	4.62	Lower
hsa-miR-1468	4.62	Lower
			hsa-miR-24	4.58	Lower
hsa-miR-181a ^*	4.56	Lower
			hsa-miR-211	4.56	Lower
hsa-let-7b	4.55	Lower
			hsa-miR-103-as	4.5	Lower
hsa-miR-302a	4.45	Lower
			hsa-miR-30b	4.45	Lower
hsa-miR-765	4.44	Lower
			hsa-miRPlus-A1031	4.43	Lower
hsa-miR-181a	4.42	Lower
			hsa-miR-25	4.38	Lower
hsa-miR-324-3p	4.37	Lower
			hsa-miR-199a-5p	4.32	Lower
hsa-miR-375	4.32	Lower
			hsa-miR-887	4.3	Lower
hsa-miR-1179	4.22	Lower
			hsa-miR-27a	4.2	Lower
hsa-miR-411 ^*	4.19	Lower
			hsa-miR-10a	4.19	Lower
hsa-miR-609	4.18	Lower
			hsa-miR-342-3p	4.18	Lower
hsa-miR-219-2-3p	4.16	Lower
			hsa-miR-299-5p	4.14	Lower
hsa-miR-1	4.14	Lower
			hsa-miR-23a ^*	4.05	Lower
hsa-miR-31	4.04	Lower
			hsa-miR-1260	4.01	Lower

hsa-miR-335	3.99	Lower
			hsa-miR-93	3.98	Lower
hsa-miR-148b ^*	3.93	Lower
			hsa-miR-376b	3.91	Lower
hsa-miR-376c	3.9	Lower
			hsa-miR-16	3.89	Lower
hsa-miR-30c	3.88	Lower
			hsa-miR-21 ^*	3.87	Lower
hsa-miR-185 ^*	3.87	Lower
			hsa-miR-139-5p	3.83	Lower
hsa-miR-331-3p	3.82	Lower
			hsa-miR-210	3.8	Lower
hsa-miR-371-3p	3.8	Lower
			hsa-miR-328	3.79	Lower
hsa-miR-886-5p	3.77	Raise
			hsa-let-7c ^*	3.77	Lower
hsa-miR-484	3.74	Lower
			hsa-miR-198	3.72	Lower
hsa-miR-584	3.72	Lower
			hsa-miR-99b ^*	3.71	Lower
hsa-miR-619	3.69	Lower
			hsa-miR-654-3p	3.66	Lower
hsa-miR-377	3.65	Lower
			hsa-miR-636	3.64	Lower
hsa-miR-921	3.63	Lower
			hsa-miR-518e ^*	3.59	Lower
SNORD49A	3.56	Lower
			hsa-miR-188-5p	3.54	Lower
hsa-miR-532-3p	3.52	Lower
			hsa-miR-1266	3.5	Lower
hsa-miR-410	3.5	Lower
			hsa-miR-34b ^*	3.5	Raise
hsa-miR-505 ^*	3.49	Lower
			hsa-miR-18a	3.49	Lower
hsa-miR-297	3.43	Lower
			hsa-miR-940	3.43	Lower

hsa-miR-582-3p	3.43	Lower
			hsa-miR-7-2 ^*	3.43	Lower
hsa-miR-30e	3.42	Lower
			hsa-miRPlus-A1027	3.42	Lower
hsa-miR-146a	3.39	Lower
			hsa-miR-21	3.38	Lower
hsa-miR-431	3.38	Lower
			hsa-miR-495	3.38	Lower
hsa-miR-106a	3.37	Lower
			hsa-miR-574-3p	3.37	Lower
hsa-miR-526b	3.37	Lower
			hsa-miR-651	3.37	Lower
hsa-miR-92a	3.37	Lower
			hsa-miR-182	3.36	Lower
hsa-miR-631	3.34	Lower
			hsa-miR-675 ^*	3.34	Lower
hsa-miR-374b ^*	3.34	Lower
			hsa-miR-300	3.31	Lower
hsa-miRPlus-C1070	3.31	Lower
			hsa-miR-135a	3.31	Lower
hsa-miR-449a	3.27	Lower
			hsa-miR-187	3.23	Lower
hsa-miR-19b-1 ^*	3.21	Lower
			hsa-miR-412	3.19	Lower
hsa-miR-345	3.18	Lower
			hsa-miR-202	3.14	Lower
hsa-miR-524-5p	3.14	Lower
			hsa-miR-10b	3.14	Lower
hsa-miR-452	3.14	Lower
			hsa-miR-141	3.13	Lower
hsa-miR-217	3.11	Lower
			hsa-miR-17 ^*	3.1	Lower
hsa-miR-200a	3.1	Lower
			hsa-miR-523	3.08	Lower
hsa-miR-642	3.07	Lower
			hsa-miR-378	3.05	Lower

hsa-miR-99b	3.04	Lower
			hsa-miR-339-3p	3.02	Lower
hsa-miR-942	3.01	Lower
			hsa-miR-555	2.99	Lower
hsa-miR-222	2.99	Lower
			hsa-miR-151-5p	2.98	Lower
hsa-miR-634	2.97	Lower
			hsa-miR-628-5p	2.96	Lower
hsa-miR-223	2.95	Lower
			hsa-miR-486-5p	2.95	Lower
hsa-miR-142-3p	2.93	Lower
			hsa-miR-130b	2.92	Lower
hsa-miR-220b	2.92	Lower
			hsa-miR-218	2.9	Lower
hsa-miR-132	2.89	Lower
			hsa-miR-20a	2.89	Lower
hsa-miR-320a	2.88	Lower
			hsa-miR-553	2.87	Lower
hsa-miR-27b	2.87	Lower
			hsa-miR-620	2.86	Lower
hsa-miR-28-5p	2.84	Lower
			hsa-miR-1913	2.83	Lower
hsa-miR-150	2.83	Lower
			hsa-miR-301b	2.83	Lower
hsa-miR-520d-3p	2.82	Raise
			hsa-miR-126	2.82	Lower
hsa-miR-654-5p	2.81	Lower
			hsa-miR-558	2.81	Lower
hsa-miR-586	2.8	Lower
			hsa-miR-516b	2.77	Lower
hsa-miR-1269	2.77	Lower
			hsa-miR-658	2.76	Lower
hsa-miR-92a-1 ^*	2.75	Lower
			hsa-miR-92a-2 ^*	2.75	Lower
hsa-miR-625 ^*	2.74	Lower
			hsa-miR-1205	2.74	Lower

hsa-miR-224 ^*	2.74	Lower
			hsa-miR-326	2.72	Lower
hsa-miR-573	2.72	Lower
			hsa-miR-1909	2.72	Lower
hsa-miR-500	2.71	Lower
			hsa-miR-7	2.71	Lower
hsa-miR-583	2.7	Lower
			hsa-miR-185	2.69	Lower
hsa-miR-943	2.69	Lower
			hsa-miR-544	2.69	Lower
hsa-miR-9	2.68	Lower
			hsa-miR-22	2.67	Lower
hsa-miR-1252	2.67	Lower
			hsa-miR-876-3p	2.64	Lower
hsa-miR-890	2.64	Lower
			hsa-miR-520c-3p	2.63	Lower
hsa-miR-1270	2.63	Lower
			hsa-miR-296-3p	2.62	Lower
hsa-miR-450b-3p	2.62	Lower
			hsa-miR-200b	2.62	Lower
hsa-miR-576-5p	2.61	Lower
			hsa-miR-767-5p	2.61	Lower
hsa-miR-888	2.61	Lower
			hsa-miR-216a	2.6	Lower
hsa-miRPlus-C1089	2.6	Lower
			hsa-miR-340 ^*	2.6	Lower
hsa-miR-214 ^*	2.59	Lower
			hsa-miR-550	2.59	Lower
hsa-miR-510	2.58	Lower
			hsa-miR-34c-3p	2.57	Lower
hsa-miR-135b	2.57	Lower
			hsa-miR-106b	2.56	Lower
hsa-miR-512-3p	2.56	Lower
			hsa-miR-1237	2.56	Lower
hsa-miR-543	2.56	Raise
			hsa-miR-18b	2.54	Lower

hsa-miR-125a-5p	2.53	Lower
			hsa-miR-135b ^*	2.53	Lower
hsa-miR-760	2.52	Raise
			hsa-miR-184	2.51	Lower
hsa-miR-629 ^*	2.47	Lower
			hsa-miR-1238	2.47	Lower
hsa-miR-138	2.47	Lower
			hsa-miR-365	2.46	Lower
hsa-let-7g ^*	2.45	Lower
			hsa-miR-744	2.45	Lower
hsa-miR-133a	2.45	Lower
			hsa-miR-557	2.44	Lower
hsa-miR-454 ^*	2.44	Lower
			hsa-miR-26b ^*	2.44	Lower
hsa-miR-593 ^*	2.43	Lower
			hsa-miR-548c-5p	2.42	Lower
hsa-miR-653	2.41	Lower
			hsa-miR-708	2.41	Lower
hsa-miR-15a ^*	2.41	Lower
			hsa-miR-452 ^*	2.4	Lower
hsa-miR-186	2.4	Lower
			hsa-miR-1972	2.39	Lower
hsa-miR-101 ^*	2.39	Lower
			hsa-miR-148a ^*	2.38	Lower
hsa-miR-548a-5p	2.37	Lower
			hsa-miR-98	2.36	Lower
hsa-miR-33a ^*	2.36	Lower
			hsa-miR-877 ^*	2.36	Lower
hsa-miRPlus-D1061	2.35	Lower
			hsa-miR-17	2.35	Lower
hsa-miR-608	2.34	Lower
			hsa-miR-92b ^*	2.34	Lower
hsa-miR-154	2.33	Lower
			hsa-miR-27b ^*	2.33	Lower
hsa-miR-93 ^*	2.33	Lower
			hsa-miR-203	2.32	Lower

hsa-miR-603	2.3	Raise
			hsa-miR-30d ^*	2.29	Lower
hsa-miR-373	2.28	Lower
			hsa-let-7f-1 ^*	2.28	Lower
hsa-miR-541 ^*	2.27	Lower
			hsa-miR-187 ^*	2.27	Lower
hsa-miR-1265	2.26	Lower
			hsa-miR-23a	2.25	Lower
hsa-miR-30c-1 ^*	2.25	Lower
			hsa-miR-362-5p	2.25	Lower
hsa-miR-30a ^*	2.25	Lower
			hsa-miR-200b ^*	2.25	Lower
hsa-miR-744 ^*	2.24	Lower
			hsa-miR-1979	2.23	Lower
hsa-let-7b ^*	2.23	Lower
			hsa-miR-132 ^*	2.23	Lower
hsa-miR-571	2.22	Lower
			hsa-miR-425 ^*	2.2	Lower
hsa-miR-194 ^*	2.2	Lower
			hsa-miR-145 ^*	2.17	Lower
hsa-miR-551b	2.17	Lower
			hsa-miR-720	2.16	Lower
hsa-miR-302d	2.16	Lower
			hsa-miR-195	2.16	Lower
hsa-miR-194	2.16	Lower
			hsa-miR-885-3p	2.16	Lower
hsa-miR-579	2.15	Lower
			hsa-miR-361-3p	2.15	Lower
hsa-miR-542-5p	2.15	Lower
			hsa-miR-320b	2.13	Lower
hsa-miR-155	2.13	Raise
			hsa-miR-548j	2.1	Lower
hsa-miR-616	2.1	Lower
			hsa-miR-502-5p	2.1	Lower
hsa-miR-662	2.09	Lower
			hsa-miR-137	2.08	Lower

hsa-miR-218-1 ^*	2.08	Lower
			hsa-miR-1537	2.07	Lower
hsa-miR-143 ^*	2.07	Lower
			hsa-miR-1227	2.06	Lower
hsa-miR-23b	2.05	Lower
			hsa-miR-675b	2.03	Lower
hsa-miR-323-3p	2.03	Lower
			hsa-miR-889	2.02	Lower
hsa-miR-485-3p	2.02	Raise
			hsa-miR-545	2.01	Raise
hsa-miR-340	2	Lower

MicroRNA in his-and-hers watches 39 carries out the non-paired t test of Benjamini and Hochberg false discovery rate (FDR)<0.05.Referring to Benjamini and Hochberg. " Controlling the false discovery rate:a practical and powerful approach to multiple testing " Journal of the Royal Statistical Society, Series B (Methodological) 57:289-300 (1995)).32 kinds of significance probes confirm to meet these standards.Referring to table 40.In table 40, the p value and the adjusting that show correction refer to the rise (rising) of comparing with the 4th group in the 3rd group and lower (decline).

Table 40:PSA<or >=multiple of the PCa sample miR level of 4.0ng/ml changes

MicroRNA	The p value	Regulate	Multiple changes
				hsa-miR-432	0.0025	Raise	31.23
hsa-miR-23b ^*	0.0073	Raise	5.09
				hsa-miR-518f	0.0073	Raise	9.76
hsa-miR-96	0.0073	Raise	14.72
				hsa-miR-154 ^*	0.0084	Raise	7.11
hsa-miR-143	0.0157	Raise	41.44
				hsa-miR-424 ^*	0.0157	Raise	5.25
hsa-miR-219-2-3p	0.0257	Raise	4.16
				hsa-miR-517a	0.0313	Raise	5.85
hsa-let-7b	0.0313	Raise	4.55
				hsa-miR-450a	0.0344	Raise	6.27
hsa-miR-204	0.0415	Raise	11.18
				hsa-miR-19b-1 ^*	0.0415	Raise	3.21
hsa-miR-217	0.0441	Raise	3.11
				hsa-miR-181a ^*	0.0441	Raise	4.56

hsa-miR-150	0.0441	Raise	2.83
				hsa-miR-629	0.0442	Raise	14.54
hsa-miR-148b ^*	0.0442	Raise	3.93
				hsa-miR-617	0.0442	Raise	4.74
hsa-miR-18a ^*	0.0442	Raise	7.24
				hsa-miR-517 ^*	0.0442	Raise	5.87
hsa-miR-451	0.0442	Raise	8.33
				hsa-miR-595	0.0442	Raise	9.96
hsa-miR-634	0.0442	Raise	2.97
				hsa-miR-93	0.0442	Raise	3.98
hsa-miR-1270	0.0442	Raise	2.63
				hsa-miR-424	0.0442	Raise	15.78
hsa-miR-299-5p	0.0442	Raise	4.14
				hsa-miR-365 ^*	0.0442	Raise	5.67
hsa-miR-215	0.0442	Raise	13.4
				hsa-miR-769-5p	0.0442	Raise	5.95
hsa-miR-1205	0.0442	Raise	2.74

In the situation that the collection of 32 kinds of probes in four above-mentioned group of 1-4 master meters 40.6 kinds of microRNAs are found to have the differential expression that surpasses 5 times, and wherein prostate cancer PSA<4.0 group mean value not with the interquartile range overlaid of described control group.These miR can have PSA<4.0 the symptom male sex is arranged in by cancer with without cancer, make a distinction.This selection is comprised of the miR shown in Figure 105 A-105F: hsa-miR-432 (Figure 105 A), hsa-miR-143 (Figure 105 B), hsa-miR-424 (Figure 105 C), hsa-miR-204 (Figure 105 D), hsa-miR-581f (Figure 105 E) and hsa-miR-451 (Figure 105 F).In described figure, X-axis shows described four sample sets." contrasting non-" is the control patients (the 2nd group) of PSA >=4.0ng/ml; " contrast is " is patient's (the 1st group) of PSA<4.0ng/ml; " ill non-" is the patients with prostate cancer (the 4th group) of PSA >=4.0ng/ml; " ill be " is the patients with prostate cancer (the 3rd group) of PSA<4.0ng/ml.

embodiment 46: the prostate cancer associated microRNA

Figure 106 shows the level of microRNA miR-29a and miR-145 the vesica that separates of plasma sample from prostate cancer (PCa) and contrast.For miR-29a, show the data of 81 example contrasts and 130 routine PCa cases.For miR-145, show the data of 81 example contrasts and 126 routine PCa cases.Paired t-test shows, the remarkable difference between case and contrast of the level of miR-29a (p<0.001) and miR-145 (p<0.0001).

embodiment 47:PCa treats the microRNA after front and treatment

15 patients with prostate cancer extract plasma sample before treatment and after treatment.Described treatment is radical prostatectomy or radiotherapy.Estimate the RNA by the microcapsule bubble acquisition of plasma sample on Exiqon microRNA instant qRT-PCR group.Further details is referring to embodiment 17-18.Result is also carried out paired t-test subsequently with respect to calibrate probe normalization method between plate.Use Benjamini and the check of Hochberg false discovery rate to proofread and correct the p value.Table 41 shows several statistically evident miR of this miR expression after and treatment front from treatment.It is the raising amount of the miR of these in sample before treatment of comparing with the sample after treatment that multiple changes.Whole miR in table 41 cross and express in sample before treatment.

Show the front miR with treating rear differential expression of 41:PCa treatment

miR	Multiple changes	The p value of proofreading and correct
			hsa-miR-1974	12.08	0.0025
hsa-miR-27b	7.8	0.0025
			hsa-miR-103	10.43	0.0067
hsa-miR-146a	9.24	0.0067
			hsa-miR-22	4.06	0.0067
hsa-miR-382	8.12	0.0105
			hsa-miR-23a	3.93	0.0181
hsa-miR-376c	3.51	0.0181
			hsa-miR-335	8.26	0.0181
hsa-miR-142-5p	3.85	0.0202
			hsa-miR-221	7.08	0.0245
hsa-miR-142-3p	3.8	0.0302
			hsa-miR-151-3p	9.1	0.0398
hsa-miR-21	3.81	0.0398

embodiment 48: vesica separation and detection method

Except method mentioned above, thereby the separation and detection that large metering method known to those of skill in the art can be used for vesica is implemented method of the present invention.The illustrative that is below several these class methods is described.

glass microballon. can be from Illumina, Inc.San Diego, CA, the VeraCode/BeadXpress that USA obtains.Step is as follows:

1. by antibody and the direct coupling of available carboxylic group are prepared to described pearl.

2. be enclosed in the lip-deep nonspecific binding site of described pearl.

3. described pearl is made an addition in vesica enriched material sample.

4. wash described sample to remove unconjugated vesica.

5. fluorescent-labeled antibody is used as detecting antibody, it should be combined specifically with vesica.

6. wash plate is to remove unconjugated detection antibody.

7. measure the fluorescence of plate hole to determine existing of vesica.

enzyme Linked Immunoadsorbent Assay (ELISA).the method of carrying out ELISA is known for those skilled in the art.Step is usually as follows:

1. preparation is surperficial, and the capture antibody of known quantity is combined thereon.

2. be enclosed in described lip-deep nonspecific binding site.

By the vesica sample application in this plate.

4. wash described plate to remove unconjugated vesica.

5. the one-level antibody that application connects as the enzyme that detects antibody, it also is combined specifically with described vesica.

6. wash described plate to remove unconjugated antibody-enzyme conjugates.

7. applied chemistry preparation, it is become color, fluorescence or electrochemical signals by described enzymatic conversion.

8. measure absorbancy, fluorescence or the electrochemical signals (as electric current) of described plate hole to determine existing and measuring of vesica.

electrochemiluminescence detects to be analyzed. can be available from Meso Scale Discovery, Gaithersburg, MD, USA:

1. by the selected damping fluid of 5mL (as PBS, TBS, HEPES) and 1%Triton X-100 (final concentration 0.015%) combination of 75 μ l are applied to damping fluid to prepare plate.

2. dilute capture antibody to be coated.

3. use plate to apply the dilution capture antibody that damping fluid (containing Triton) prepares every hole 5 μ l.

4. the capture antibody of 5 μ l dilutions is directly applied to the center of working electrode surface, note not destroying dielectric medium.Small droplets should diffuse in time the edge of dielectric barrier but not cross described edge.

5. make plate not cover and non-standing over night intrusively.

The solution that will comprise the sample of vesica and comprise marker detection antibody adds to described plate hole.Described detection antibody is the anti-target antibody with electrochemiluminescence compound MSD SULFO-TAG marker mark.Be present in that the capture antibody of vesica on being fixed in electrode in sample is combined and the target of described marker detection antibody on described vesica is combined, thereby completed sandwich form (sandwich).Add MSD playback buffer liquid to provide electrochemiluminescence to detect necessary environment.Plate is inserted and to read, in the plate instrument, wherein voltage to be put on plate electrode, and this marker that causes being incorporated into electrode surface is luminous.Read the plate instrument and detect light emitted intensity so that the quantitative measurment to vesica amount in described sample to be provided.

nano particle. organize gold nano grain more and use the independent antibody of being combined with each particle to be prepared.On slide, concentrated microcapsule bubble and single pearl type are hatched 4 hours under 37 ℃.If there are enough targets, the chroma offset from redness to purple appears.Each target is carried out to described analysis independently.Gold nano grain can be available from Nanosphere, Inc., Northbrook, Illinois, USA.

nanosight. can use the detection of particles of optics to measure the diameter of one or more vesicas.Referring to the United States Patent (USP) 7,751,053 that is entitled as " Optical Detection and Analysis of Particles " of authorizing on July 6th, 2010; And the United States Patent (USP) 7,399,600 that is entitled as " Optical Detection and Analysis of Particles " of mandate on July 15th, 2010.But the described particle of mark it is counted also, thereby can assess the amount of different vesicas in sample or vesica colony.

embodiment 49: the scheme of concentrating vesicles from blood plasma

In the present embodiment, concentrating vesicles in patient's plasma sample.Described scheme can be used for the vesica analysis of patient's sample, and it comprises the detection of vesica surface biological mark (as surface antigen) as described herein and/or vesica useful load (as mRNA and microRNA).

equipment, reagent and supply:

equipment

A.Thermo Scientific Sorvall Legend RT Plus series desktop whizzer, be equipped with the rotary head of 15ml well-bucket.Unit number: 75004377 (Thermo Scientific, the branch of Thermo Fisher Scientific, Waltham, MA)

B. the II level Biohazard Safety Equipment operated for blood plasma

C. transfer pipet manager: 20 μ l, 200 μ l, 1000 μ l

D. serum pipette manager, Pipette Boy, VWR, numbering: 14222-180 (VWR International, LLC, West Chester, Pennsylvania)

E.VWR numeral turbine mixer, numbering: 14005-824

F. the computer that there is the internet access

reagent

a.1×PBS，Sigma?pH?7.4。Numbering: P-3813 (Sigma, Saint Louis, Missouri, the branch of Sigma-Aldrich, Inc.)

B. molecular biology reagent water, Sigma, numbering: W4502

supply

A.0.8 μ m Millex-AA injector drive type filtration unit, Millipore.Unit number: SLAA033SB (Millipore, Billerica, MA)

The b.Pierce thickener, 150K MWCO (weight shutoff value), 7ml.Unit number: 89922 (Pierce, the branch of Thermo Fisher Scientific Inc., Rockford, IL)

C. the BD Luer Lock syringe of non-sterilizing, 10ml, unit number: 301029 (BD, Franklin Lakes, NJ)

The non-binding pipe of d.USA Scientific copolymer 1 .5ml, USA Scientific, numbering 1415-2500 (USA Scientific, Inc., Ocala, FL)

E.5ml aseptic insert serum pipette, Fisher, numbering 13-678-11D (Fisher Scientific, the branch of Thermo Fisher Scientific, Pittsburgh, PA)

F. ice bucket, Fisher, numbering 02-591-46

G. test-tube stand, Fisher, numbering 05-541-38

H. four-way frame, Fisher, numbering 03-448-17

I.50ml bore bottom tube, VWR, numbering 21008-951

J. float type test-tube stand, VWR, numbering 60986-100

K.1 rise beaker, VWR, numbering 89000-226

L.10/20 μ l filters liquid-transfering sucker, Rainin, numbering GP-L10F (Rainin Instrument, LLC, Oakland, CA, a METTLER TOLEDO Company)

M.200 μ l filters liquid-transfering sucker, Rainin, numbering GP-L200F

N.1000 μ l filters liquid-transfering sucker, Rainin, numbering GP-L1000F

O. personal protective device

quality control:

A. the sample had lower than 900 μ l volumes may provide the result of suboptimal and should avoid.

B. carried out the result of suboptimal may being provided and should avoiding over the sample of a freeze-thaw cycle.

C. described 150K MWCO post may be subject to the damage of liquid-transfering sucker or sustain damage during manufacture.Can assess definite that post damages by the check permeate.Comprise if permeate shows the blood plasma (<100 μ l) that a large amount of blood plasma and described post self comprise low volume, probably this 7ml 150K MWCO post is damaged.If suspect that post is damaged, sample need to be used another blood plasma aliquots containig from same patient again to concentrate.

process:

select for concentrated sample

A. will select from obtaining of sample data storehouse the sample message input Microsoft excel spreadsheet list (Microsoft Corp, Redmond, WA) of sample.

B. print a plasma extraction worksheet (Plasma Concentration Bench Sheet) from Excel.

filtration procedure to plasma sample

A. by the water filled with in ice chest from the cold running water tap.

B. find the plasma sample of listing on the plasma extraction worksheet and take out from-80 ℃ of (65 ℃ to-85 ℃) refrigerators.Any residue cryopreservation tube is stored in continuing in the identical box of-80 ℃ (65 ℃ to-85 ℃) needs the situation of other blood plasma aliquots containig for test with reply.

C. by inserting in the float type test-tube stand and melt in the water that sample is extracted in a).Inspected sample after 10 minutes, and in the situation that all plasma sample melt fully to keep somewhere in water by blood plasma and take and within 5 minutes, inspected as interval until all plasma sample thawings.

D. during thawing step, take out label and each 7ml 150K MWCO post (one of every plasma sample) is attached one piece of side label and is placed in the four-way test-tube stand from blue folder.The lot number of post is recorded in the plasma extraction worksheet.

E. the molecular biology reagent water is poured in the beaker of 1 liter.

F. for each sample to be moved, by syringe tip being immersed in the water in beaker and twitching inner core and the 10ml syringe is filled to the molecular biology reagent water with 4ml.

G. the Millipore filter of 0.8 μ m is attached to the most advanced and sophisticated of each syringe and inclusion is arrived on described 7ml 150K MWCO post by described filter.

H. described post is added a cover, and is placed in described bucket centrifuge and descends centrifugal 4 minutes at 20 ℃ (16 ℃ to 24 ℃) at Sorvall Legend XRT desk centrifuge with 1000 * g.

I. carry out centrifugal the time when coupled columns, from syringe, remove the Millipore filter, inner core is extracted to injection and filter is placed in to injector tip again.

J. complete when centrifugal when whizzer, discard the percolation thing of described 7ml 150K MWCO post and stay the residual water-content in the filter of top.

K. syringe and filter are placed on open 7ml 150K MWCO post.The opening end of syringe is filled to the 1 * PBS (it prepares in aseptic molecular level water) with 5.2ml.

L. by using the p1000 transfer pipet, assess patient blood plasma volume and it is recorded on the plasma extraction worksheet.

If m. sample is less than 900 μ l, tackle the new test that this patient carries out another blood plasma aliquots containig.Obtain another sample of described patient and correspondingly upgrade the plasma extraction worksheet.

N. patient's blood plasma (900-1000 μ l) is moved in the PBS of described syringe, mix twice with pipettor, and blood plasma pipe and any residual patient's blood plasma are discarded in the Biosafety trash receptacle together with liquid-transfering sucker.

O. inner core is inserted in syringe and slowly and oppress this inner core until the content of described syringe arrives on described 7ml 150K MWCO post by filter (～1ml/ second).

P. make whole sample by filter until described 7ml 150K MWCO post is full of liquid or can sees foam by described filter.

Q. syringe and the filter attached are discarded in the Biosafety trash receptacle and closely cover all 7ml 150K MWCO posts.

Attention: step k)-q) should carry out in Biohazard Safety Equipment.

Attention: if the percolation thing of blood plasma is unlimpid and in concentrated any point generation variable color, likely post is damaged and should abandon described sample.Similarly, if concentrated blood plasma volume is down to below 100 μ l at any point between diakinesis, should abandon described sample.In either case, the plasma sample that look for novelty and repeat this process.

vesica concentrates centrifugal scheme

A. under 20 ℃ (16 ℃ to 24 ℃) with the centrifugal 7ml 150K of 2000 * g MWCO post 1 hour.Open whizzer and inspect sample to check whether it is down to the long-pending scope of following plasma extraction object:

Target volume: 0.3 * primitive plasma volume (μ l)

The minimum volume that allows: 100 μ l

For example: if the primitive plasma volume is 900 μ l, target volume should be 270 μ l (0.3 * 900=270).

B. 1 hour centrifugal during, in 1 liter of beaker the preparation 100ml 10% sodium hypochlorite solution.

C. 1 hour centrifugal during, each copolymer 1 .5ml pipe (one, every sample) is attached to one piece of side label.

D. after 1 hour centrifugal, the percolation thing is poured in 10% sodium hypochlorite solution.When beaker is full of or all sample has been poured out, pour into liquid discharge pipe.

E. visual inspection sample volume.If the plasma extraction thing surpasses the 8.5ml scale of evaporating pipe, continue with the increment of 10 minutes under 20 ℃ (16 ℃ to 24 ℃) with the centrifugal plasma sample of 2000 * g, inspect volume after each centrifugal, until the plasma extraction thing is 8.0 to 8.5ml.

F. avoid the liquid-transfering sucker white filter of swiping during this step.When described centrifugal end, inhale lentamente to beat on described post with the p1000 pipettor that is set as 150 μ l and mix at least 6 times (avoiding producing bubble), and it is long-pending to determine the plasma extraction object to regulate pipettor.As fruit volume, between 100 μ l and target volume, concentrated blood plasma is transferred in the copolymer 1 .5ml pipe of mark before this.As fruit volume still higher than target volume, repeating step e).

G. record concentrated blood plasma volume on described plasma extraction worksheet, and evaporating column is discarded in the Biosafety trash receptacle.

H. input described blood plasma volume, enriched material volume, thickener lot number in electronics plasma extraction worksheet, preserve, print a new worksheet and it is attached on original a worksheet.

I. will in aseptic molecular level water, prepare～1 * PBS of 45ml pours in 50ml cone bottom tube with for next step.

J. according to the plasma extraction worksheet of above printing, the 1 * PBS that adds appropriate amount with by described sample reconstruct to reach target volume.

K. before operating analysis next day, under 4 ℃ (2 ℃ to 8 ℃) by concentrated plasma sample store overnight on test-tube stand.With plastic plug covering pipe support and to plug mark date and accession number.

Calculate:

A. the final volume x=y of concentrated plasma sample * 0.3, wherein x is that final volume and the y of enriched material are initial blood plasma volumes.

Example: sample volume is 900 μ l.900 μ l * 0.3=270 μ l final volume.

reference:

The a.Pierce thickener, 150K MWCO (weight shutoff value), 7ml.Unit number: 89922 product insets.

embodiment 50: the microballoon vesica of concentrated blood plasma is analyzed

This embodiment has provided for estimating the method for the concentrated patient's plasma sample of concentrating vesicles.This scheme can be used for the analysis to the vesica surface biological mark in the deshydremia slurry samples according to embodiment 49 described processing.

equipment, reagent and supply:

equipment

A.VWR numeral turbine mixer, numbering 14005-824 (VWR International, LLC, West Chester, Pennsylvania)

B.Boekel Scientific Jitterbug 4, number 270440 (Boekel Scientific, Feasterville, PA)

C.Pall life sciences vacuum manifold, number 13157 (Pall Corporation, East Hills, New York)

D.Pall life sciences porous flat plate vacuum manifold, number 5017

E.Pall life sciences 1ml dash receiver spacing block (receiver plate spacer block), number 5014

F.Pall life sciences waste liquid is discharged adaptive receptacle (waste drain adapter retainer), numbers 5028

G. single passage transfer pipet manager: 2 μ l, 10 μ l, 20 μ l, 200 μ l, 1000 μ l

H.8 passage transfer pipet manager: 20 μ l, 200 μ l

I. electronics 8 passage pipettors: 1000 μ l

J. electronics single passage pipettor: 200 μ l, 1000 μ l

K. serum pipette manager, Pipette Boy, VWR, numbering 14222-180

L.Luminex LX200 instrument (Luminex Corporation, Austin, TX)

M. microplate vibrator, VWR, numbering 12620-926

N.VWR MiniFuge microcentrifuge, VWR, numbering 93000-196

O. ice-making machine, Scotsman, numbering AFE424 (Scotsman Ice Systems, Vernon Hills, IL)

reagent

A. note: hereinafter listed antibody reagent is exemplary antibodies.Tested for the antibody of catching and/or detect to substitute, selected as required the antibody for described target organism mark.

Exemplary acquisition antibody-selected according to required test target.Referring to table 42.

Table 42: capture antibody

Microplex microballoon with coupling antibody. capture antibody is coupled to required microballoon, and it is selected from the carboxylation MicroPlex of fluorescent dye microballoon pearl, SeroMAP ^tMmicroballoon pearl and MagPlex microballoon pearl (Luminex Corporation, Austin, TX).The scheme of using manufacturers to provide is carried out coupling.Referring to " SAMPLE PROTOCOL FOR TWO-STEP CARBODIIMIDE COUPLING OF PROTEIN TO CARBOXYLATED MICROSPHERES ", " SAMPLE PROTOCOL FOR CONFIRMATION OF ANTIBODY COUPLING " and the relevant programme that can obtain online on www.luminexcorp.com/support/protocols/protein.html.Further details is provided in this paper embodiment.

Exemplary conjugate is hereinafter shown in table 43.Suitable arbitrarily antibody can be used for to coupling, as, any in the antibody of above listing in table 42 or target are in other antibody of target antigen.

Table 43: microballoon coupling antibody

^*the information of coupling antibody is found in the capture antibody of table.

Detect antibody one and can use various markers.Show the exemplary antibodies for four transmembrane protein CD9, CD63 and CD81.Can use as required for other biomarker as the antibody of vesica biomarker, cell source specific biological mark or disease biomarker.Referring to table 44.

Table 44: detect antibody

^*phycoerythrin

A. contain the phosphate buffered saline (PBS) (PBS) of BSA, pH7.4, Sigma, catalog number P3688-10PAK (Sigma, Saint Louis, Missouri, Sigma-Aldrich, the branch of Inc.)

Starting block sealing damping fluid in b.PBS, Thermo Scientific, catalog number 37538 (Thermo Scientific, the branch of Thermo Fisher Scientific, Waltham, MA).

C.PBS-BN (pH 7.4 for PBS, 1%BSA, and Sigma numbers P3688,0.05% sodiumazide, Sigma, catalog number S8032)

D. aseptic molecular level water (without DNA enzyme and RNA enzyme, 0.1 μ m filter), Sigma, catalog number W4502

E.VCaP microcapsule bubble (2.14 μ g/ μ l)

F. normal male blood plasma, lot number #55-24482-042610 (Innovative Research, sample 55-24482), create for the VCaP contrast

supply

A.USA Scientific Temp Assure PCR 8-pipe row bar, numbering 1402-2908 (USA Scientific, Inc., Ocala, FL)

B.Millipore Multiscreen HV Luminex screen plate, 0.45 micro-M, transparent, vinylbenzene, Millipore catalog number MSBVN1250 (Millipore, Billerica, MA)

The non-binding pipe of c.USA Scientific copolymer 1 .5ml, catalog number 1415-2500

D.USA Scientific TempPlate sealed foil, catalog number 2923-0110

E. disposable filtering and aseptic liquid-transfering sucker, without DNA enzyme, RNA enzyme and pyrogen

F.1L vial, VWR, catalog number 89000-240 (VWR International, LLC, West Chester, Pennsylvania)

G.250ml vial, VWR, catalog number 89000-236

H. stirring rod, VWR, catalog number 58948-218

I. ice bucket, Fisher, catalog number 02-591-46 (Fisher Scientific, the branch of Thermo FisherScientific, Pittsburgh, PA)

J.96 hole Falcon flat board, VWR, catalog number 62406-321

K.1L measuring graduates, Fisher, catalog number 03-007-36

L. grillage, Fisher, catalog number 05-541-55

M. test-tube stand, Fisher, catalog number 05-541-38

N. four-way frame, Fisher, catalog number 03-448-17

O.15ml bore bottom tube, VWR, catalog number 21008-918

P. reagent storage box, VWR, catalog number 89094-662

Q.10/20 μ l filters liquid-transfering sucker, Rainin, catalog number GP-L10F (Rainin Instrument, LLC, Oakland, CA, METTLER TOLEDO Company)

R.200 μ l filters liquid-transfering sucker, Rainin, catalog number GP-L200F

S.1000 μ l filters liquid-transfering sucker, Rainin, catalog number GP-L1000F

T.1000 the non-filtration liquid-transfering sucker of μ l, Rainin, catalog number GP-L1000

U. aluminium foil, Fisher, 01-231-100

V. personal protective device

W. Major program template electrical form (Master Plan Template Spreadsheet) (tracking electrical form)

quality control:

analysis of control

Analysis of control is comprised of the microcapsule bubble from described VCaP clone.VCaP shifts by the vertebrae that comes from the 59 years old Caucasia male patient who suffers from hormone refractory type prostate cancer the human epithelial cell system that thing is set up in 1997.It goes down to posterity as heterograft in mouse, subsequently vitro culture.Described VCaP cell be the male sex hormone sensitivity and produce vesica.Referring to Korenchuk, S. etc., VCaP, a cell-based model system of human prostate cancer.In Vivo, 2001.15 (2): p.163-68; Jansen, F.H. etc., Exosomal secretion of cytoplasmic prostate cancer xenograft-derived proteins.Mol Cell Proteomics, 2009.8 (6): p.1192-205.

Move in triplicate VCaP microcapsule bubble (MVS) height and blank group to confirm (1) pearl master mixture performance on each plate, the technical specification of (2) single operation, and (3) detect the antibody performance.The high contrast of described VCaP MVS is comprised of the VCaP microcapsule bubble (it is diluted in normal male blood plasma) of 0.5mg/ml purifying.The VCaP microcapsule bubble of the 0mg/ml purifying of described VCaP MVS blank in the PBS background microcapsule bubble of purifying (that is, without) forms.

If average signal (high VCaP MVS contrast) is higher than at least 10 times of backgrounds with the ratio of average background (blank VCaP MVS contrasts), think effective operation.The signal of this magnitude shows that the pearl that catches of each coupling has sufficient sample binding ability and carried out technical complete operation.

If move and do not meet the criterion of setting up for described VCaP MVS contrast, repeat whole service.The operation repeated is formed (referring to the plasma concentration of embodiment above) by patient's blood plasma of described VCaP MVS contrast and another plasma concentration.According to received number of samples, described operation is repeated maximum twice.If operate in last failure, by described sampling report for not obtaining effective result failure.

internal contrast

Four transmembrane protein capture antibodies (CD9, CD63 and CD81) are as the internal contrast of the adequacy of each specimen.Calculate average MFI (meta fluorescence intensity) for three kind of four transmembrane protein capture antibody.If described average comprehensive MFI value surpasses 500, think that described sample has sufficient microcapsule bubble concentration for further test.

If sample does not also meet the criterion of setting up for described four transmembrane protein capture antibodies, retest described sample.The operation repeated is formed (referring to the plasma concentration of embodiment above) by patient's blood plasma of VCaP MVS and another plasma concentration.If there is the other aliquots containig of patient's blood plasma, repeat at most again twice of described operation.If that repeats operates in last failure, described sample is reported as and does not obtain the non-appreciable of effective result.

restriction:

If collect improperly or the store patient sample, the vesica here may be degraded or its protein content changes.The degraded of vesica may cause assembling and wrong protein expression result output, thereby produces indefinite or wrong result.

process:

Except washing step, use in steps the filtering type suction nozzle of transfer pipet.

A. open suitable Major program template electrical form and above suitable information inserted in the yellow unit of any blank in batch information bar (Lot Info tab).Preserve described document.

B. select described worktable wall scroll (Work Bench Sheet tab) (tracking and guiding work table in described Major program template electrical form) and print a hard copy for using on table top.

C. take out the deshydremia slurry samples of the day before yesterday and be placed on worktable from refrigerator.Referring to the embodiment that above prepared by concentrated blood plasma.

D. prepare VCaP MVS contrast according to the formula of worktable list.

I. insert trash ice in ice bucket.

Ii. each plate is taken out from-80 ℃ (65 ℃ to-85 ℃) that 1 pipe VCaP MVS converges thing (control sample converged) and 1 pipe normal serum and in thawing on ice.

Iii. converge 10 seconds of thing with 1600rpm vortex VCaP MVS.

Iv. the VCaP contrast (with reference to the worktable list) for preparing 0.5 μ g/ μ l.The normal plasma that the normal plasma pipe comprises 11.1 μ l, the VCaP that the VCaP pipe comprises 5 μ l.The VCaP MVS of appropriate amount is converged to thing (orange pipe) to be moved into normal plasma pipe (purple pipe) and mixes 5 times with transfer pipet.

V. pipe be placed on grillage and hatch 1 hour at Jitterbug with the 550rpm vibration under 37 ℃.

E. when hatching contrast, prepare sample pearl mixture.

I. by new 1.5ml copolymerization property management marked with date, initial and " sample pearl mixture ".

Ii. starting block and sample pearl mixture are added in the pipe of mark (with reference to the worktable list).

Iii. on VWR numeral turbine mixer with 5 seconds of 1600rpm vortex.

Iv. wrap up and hatch at least 10min with aluminium foil on worktable.

F. when hatching contrast, according to the plate figure on the worktable list, from storing, grow the 8-pipe row bar that takes out necessary number container.Each is arranged bar and represents the row of 1 on plate figure (in every plate, the maximum number of 8-pipe row bar is 12).

G. on grillage, 8 pipes rows bars are carried out arranged vertically and add a cover for each pipe every row.To start at upper left quarter 1 to pipe jacking row labels and from pushing up the end of to subsequently serial number from left to right.For example, row bar 1-6 in Figure 107 mark with 1-48.Row bar 7-12 on the second grillage mark with 49-96.

H. according to the plate figure on described worktable list, each deshydremia slurry samples of 50 μ l is transferred in described 8-pipe row bar in triplicate.

I. open the whole pipes except 1,2,9,10,17 and No. 18 (these pipes are preserved for the high contrast of VCaP and blank).

Ii. on digital turbine mixer with at least 5 seconds of each deshydremia slurry samples pipe of 1600rpm vortex, thereby before sample being divided into to 8-pipe row bar abundant pooled plasma.

Iii. use the p200 pipettor, sample is transferred to 8-pipe row bar, blanked-off pipe lid after each interpolation sample.

1. by drawing the concentrated blood plasma of 50 μ l in liquid-transfering sucker and disposable distribution counter sample QC and each new liquid-transfering sucker is carried out to pre-wetted.

2. use identical liquid-transfering sucker draw the concentrated blood plasma of other 50 μ l and be dispensed in correct pipe, guarantee liquid-transfering sucker to be positioned to the bottom of this pipe in a minute timing.Liquid-transfering sucker is shifted out straight in managing, carefully liquid-transfering sucker was not dragged to the pipe side.

3. repeat above-mentioned steps 2) until comprising 50 μ l, whole three pipes of this sample concentrate blood plasma twice.Identical suction nozzle can be used for whole three aliquots containigs of same sample, but different sample rooms should be changed suction nozzle.

I. after the VCaP contrast of carrying out 1 hour is hatched, use the p20 pipettor that the 0.5 μ g/ μ l VCaP contrast of 4 μ l is moved in

pipe

1,9 and 17.

J. 1 * PBS of 4 μ l is moved in

pipe

2,10 and 18.

K. the pearl mixture will be entered in all samples.

I. all pipes are opened.

Ii. on VWR numeral turbine mixer with 5 seconds of 1600rpm vortex sample pearl mixture.Repeat this vortex step before each pipette, extract in succession.

Iii. use 200 μ l electronics repetitive pipettors to add the pearl mixture of 4 μ l to each sample hose (comprising control tube), blanked-off pipe lid after each interpolation pearl.

Iv. on the mini-galaxy whizzer, 8 pipes rows bars are carried out to 1 second quick centrifugal with by whole liquid set in the bottom of described pipe.

V. in the Jitterbug of 37 ℃ (35 ℃ to 39 ℃), with 550rpm, hatch 2 hours.

Vi. can be wrapped in aluminium foil by any excessive pearl and remain under 4 ℃ (2 ℃ to 8 ℃) and spend the night and using in next day as excess thing (overage).This usage of residue pearl can be continuous one week, but any outmoded pearl should be abandoned in the Biosafety trash receptacle on every Mondays.

L. between 2 hour incubation period, prepare detection agent antibody.

I. 15ml is bored to bottom tube marked with date, initial and " detection antibody ".

Ii. add PBS-BN (volume is with reference to the worktable list) in 15ml cone bottom tube.

Iii. add CD9, CD81 and CD63 (volume is with reference to the worktable list) in PBS-BN.

Iv. on VWR numeral turbine mixer with 5 seconds of 1600rpm vortex.

V. be wrapped in aluminium foil and be placed in the four-way frame until use.

M. PBS-BN is filled in disposable storage box.

If n. be less than onboard 23 samples, by scissors cutting aluminium foil sealer, with the sky that covers any empty row and adhere on plate, list.

O. during following steps, liquid-transfering sucker should never touch the bottom of filtering plate hole.All the time with the sidepiece of suction nozzle contact hole.In addition, vacuum should be all the time moved between 3 inches and 5 inches of mercury.Plate should be only during drawing on vacuum manifold; In all other step process, plate should be placed on worktable.

P. use 1000 μ l electronics Multi-channel liquid transfer devices and 1000 type non-filtering suction nozzles with the PBS-BN pre-wetted 1.2 μ m Millipore screen plates in 100 μ l/ holes and aspirate by vacuum manifold.

Q. plate before removing, manifold is being pressed to vacuum release button.Blot the plate bottom on clean paper handkerchief.

R. use the p200 Multi-channel liquid transfer device PBS-BN of 150 μ l to be added to each hole of described plate.

S. after 2 hours hatch, sample is removed and to carry out for 1 second in the mini-galaxy whizzer centrifugal fast from Jitterbug.

T. according to the plate figure on described worktable list, the sample through hatching is transferred in the Millipore screen plate.

I. onboard from left to right (1-12 row) carry out taking out one 8 pipe row's bar and being placed in the hollow plate frame from grillage is last.Write the numbering that covers in pipe and confirm that in suitable order and direction 8-pipe row bar is used in chronological order by examining again.

Ii. use the p20 multi-channel micropipettor that two contrasts in described 8-pipe row bar are transferred in the appropriate well of screen plate.During aspirating with pipettor mix 5 times with guarantee whole pearls before being dispensed into screen plate in solution, in PBS-BN, with pipettor, mix twice.

Iii. use the p200 Multi-channel liquid transfer device that whole blood plasma is transferred to screen plate.

Iv. concentrated blood plasma possibility extreme thickness, therefore slowly mix each sample 5 times with micropipet, to guarantee blood plasma, in liquid-transfering sucker, moves up and down.If there is sample not move, increase number of times that pipettor mixes until each sample has mixed at least 5 times.Sample is transferred to screen plate, mixes twice with pipettor in PBD-BN.

V. after each 8-pipe row bar sky, will arrange bar be discarded into the Biosafety trash receptacle before confirmation all the elements thing all remove.

If vi. in any pipe, retain any liquid, repeat above step I)-iv).

Vii. continue above step I)-v) until whole sample has been added into screen plate.

If arbitrary hole, any some place u. in following step process bonding still is sucked in other hole, continue 5 seconds of constant vacuum, if some samples still bond and do not move through described filter, this hole of mark on (with sign) and described worktable list on described plate.Using the p1000 pipettor that the liquid suction is portalled and keep this hole during all steps in succession is sky.

V. aspirate lentamente supernatant liquor by vacuum manifold.Plate before removing, manifold is being pressed to vacuum release button.Blot the plate bottom on clean paper handkerchief.

W. use p1000 electronics Multi-channel liquid transfer device and 1000 μ l type non-filtering suction nozzles, with the PBS-BN of 200 μ l, wash each hole, aspirate, press vacuum release button, remove plate and blot up hill and dale the plate bottom on clean paper handkerchief from manifold subsequently.

X. repeat abovementioned steps to be amounted to 2 washings, 200 μ l PBS-BN are used in each washing.

Y. use the p200 Multi-channel liquid transfer device that the PBS-BN of 50 μ l is added in each hole.

Z. use p1000 electronics single passage pipettor to add the detection antibody (from above) of 50 μ l dilutions to each hole.

I. any excessive detection antibody can be wrapped in aluminium foil and in 4 ℃ (2 ℃ to 8 ℃) lower preservation and spend the night and using in next day as the excess thing.This usage of residue detection antibody can be continuous one week, but any old detection antibody should abandon in the Biosafety trash receptacle.

Aa. use the plate sealed foil to cover described screen plate.Gently along the periphery sealed foil, carefully in hole, do not produce malleation, because this will force liquid beyond the filter bottom.

Bb. under 25 ℃ (22 ℃ to 27 ℃), with 550rpm, on Jitterbug, hatch 1 hour.

Cc. between the incubation period of 1 hour, carry out all necessary maintenance and/or calibration with reference to Luminex Maintenance and Calibration SOP (MA-25-0009) and on Luminex pearl reading apparatus (bead reader machine).

Dd. after safeguarding and/or having calibrated, tablet information in Luminex software.

I. opening xPONENT 3.1 softwares and login enters.

Ii. click the Batches bar.

● under Batch Name, input is still added extra underscore and 1 at the end of near the plate ID at worktable list top.

1.ID：20100915_SampleV1_PME_1

Iii. its after mean to enter the numbering of uploading of data storing.For example:

1.Batch?Name：20100915_SampleV1_PME_1_1

Iv. click Create a New Batch from an Existing Protocol and select required scheme.

V. click Next.

Vi. highlight by plate figure, clicking and pull the hole that comprises sample and contrast.

Vii. click the Unknown button of plate figure below.

Viii. click Import List button on the screen right side and navigate to suitable derivation text, to its selection, clicking subsequently Open.

Ee. after the sample of 1 hour is hatched, from Jitterbug, remove screen plate, remove the paper tinsel sealer and aspirate supernatant liquor with vacuum manifold.Press vacuum release button, subsequently plate is removed from manifold, and blot up hill and dale the plate bottom on clean paper handkerchief.

Ff. use p1000 electronics Multi-channel liquid transfer device and 1000 μ l type non-filtering suction nozzles to wash each hole, aspirate, press vacuum release button with the PBS-BN of 100 μ l, remove plate and blot up hill and dale the plate bottom on clean paper handkerchief from manifold subsequently.

Gg. repeat abovementioned steps to be amounted to 2 washings, 100 μ l PBS-BN are used in each washing.

Hh. use the p200 Multi-channel liquid transfer device that the PBS-BN of 100 μ l is added to each hole.

Ii. use the plate sealed foil to cover screen plate.Gently along periphery, seal described paper tinsel.

Jj. flat board is placed in to upper 20 minute of VWR microplate vibrator of 950rpm.On Luminex 200 instrument, analyze dull and stereotyped.

I. take out plate and remove the paper tinsel sealer from vibrator.

Ii. click the Eject button of described bottom of screen.

Iii. flat board is placed in to pulling box (A1 enters the upper left corner).

Iv. click the Retract button of bottom of screen.

V. click the Run Batch button in the screen lower right corner.

Vi. hit OK in pop-up window.

Kk. when end of run, go to the Result bar, choose the Saved Batches in left side, the Exp Results of high aobvious described operation and click bottom of screen is to derive the .csv file.

Ll. store described .csv file to suitable webserver position.

Mm. login enters data analysis software, goes to Lab Queues bar, and chooses near the Import Results upper right corner.

Nn. click Browse and navigate to the .csv be positioned in clinical driving, and click Open.

Oo. confirm data in data analysis software with from Luminex 200 instrument, export consistent.

embodiment 51: prostate cancer (PCa) the vesica analysis of using multiple analysis to carry out

In the present embodiment, according to the overall process analysis of describing in embodiment 27 from suffering from prostate cancer (PCa) or not suffering from the patient's of PCa (normally) plasma sample.Prepare blood plasma and carry out multiple analysis according to described in embodiment 50 according to the scheme of embodiment 49.Capture antibody for the vesica surface antigen protein in table 45 is screened for the biomarker to detecting PCa.

Table 45:PCa capture antibody

According to shown in table 45, according to operability and according to required testing needle the Multiple Antibodies to single target organisms mark.This allows to determine and anyly as detecting PCa, provide required performance using different surfaces epi-position assessment vesica to catch, take.

embodiment 52: colorectal carcinoma (CRC) the vesica analysis of using multiple analysis to carry out

In the present embodiment, according to the overall process analysis of describing in embodiment 27 from suffering from colorectal carcinoma (CRC) or not suffering from the patient's of CRC (normally) plasma sample.Prepare blood plasma and carry out multiple analysis according to embodiment 50 according to the scheme of embodiment 49.Capture antibody for described vesica surface antigen protein in table 46 is screened for the biomarker to detecting CRC.

Table 46:CRC capture antibody

Shown according to table 46, according to operability and according to required testing needle the Multiple Antibodies to single target organisms mark.This allows assessment to use the vesica of different surfaces epi-position to catch, so that any required performance that provides to be provided.

Figure 108 A shows and uses several vesicas of catching biomarker and identifying to change multiples, and the described biomarker of catching detects to compare with normal person cross the vesica biomarker of expressing in CRC.The 128 routine samples altogether that use is comprised of 49 normal persons, 20 persons of obscuring and 59 CRC are implemented the CRC that the antibody capture by vesica carries out and are detected.Obscure sample comprise there is rheumatoid arthritis, the sample of asthma, diabetes, bladder cell carcinoma, renal cell carcinoma and chronic or acute diverticulitis.In the CRC sample, 16 examples are the I phase, and 19 examples are the II phase, and 24 examples are the III phase.As directed, the biomarker repeated in described list is the different antibodies for same antigen.Figure 108 A shows and uses for the antibody capture of multiple biomarker and detect vesica and the differentiation to normal and CRC sample carried out.Capture antibody illustrates on X-axis, and detection antibody illustrates on Y-axis.Demonstrate the maximum antibody improved and comprise the antibody for CD66 (CEA), A33, EPHA2, TROP2, DR3, UNC93A, NGAL and MUC17 in the cancer sample.Hereinafter table 47 shows sensitivity and the specificity of using various capture antibodies to obtain:

Table 47: use the antibody capture of vesica and the CRC that carries out detects

Mark	Sensitivity	Specificity
			DR3	82％	86％
STEAP
		100％	71％
epha2
		90％	83％
TMEM211
		100％	84％
unc93A				86％	84％
	A33	100％	75％
CD24				98％	77％
	NGAL
		94％	81％
	EpCam
		90％	62％
	MUC17			86％	77％
TROP2		96％	80％
	TETS			86％	80％

Figure 108 B shows the result from similar experiment, and difference is that Y-axis is the meta fluorescence intensity (MFI) in CRC and normal specimens as shown in legend.Use the second sample sets of 10 routine CRC samples and 10 routine normal specimens to repeat the experiment shown in Figure 108 B.Result is illustrated in Figure 108 C.Confirming as the mark that is bigger than expression most between cancer and normal person is similar for using arbitrary sample sets.Figure 108 D shows the various combinations of the above-mentioned mark of use to distinguish the ability of normal and CRC.Described figure shows the MFI of marked mark on X and Y-axis.CD24 is used as the colon mark, and TROP2 is as cancer markers, and four transmembrane protein CD9, CD63 and CD81 are general vesica marks.

The ability of analyzing the MFI of kinds of surface antigen in single multiplex experiment can be used for finding optimum target organisms mark.Identical technology can be applicable to various situation (as, various disease, various cancers, different target biomarker, diagnosis, prognosis or treatment diagnosis etc.) to identify that the new bio mark is for analysis exploitation subsequently.

embodiment 53: use TMEM211 and the CD24 detection to colorectal carcinoma (CRC)

According to described herein on the microballoon platform the concentrated microcapsule bubble plasma sample of operation.To with pearl, be connected for the antibody of various surface antigens and for catching microcapsule bubble.Several antibody demonstrates significant difference between the sample from CRC and normal patient.Use the microcapsule bubble of CD9, CD63 and/or CD81 mark capturing.In the present embodiment, use and catch from the vesica in sample (Figure 109 A-H) for the capture antibody of TMEM211 and/or CD24.According to the method described in embodiment 52, implement to analyze.

CD24 is the fixed albumen of glycosyl-phosphatidyl inositol anchor (Pierres etc., 1987; Kay etc., 1990; Alterman etc., 1990), it expresses on the immature cell of the main hematopoietic cell lineage of great majority (if not all) and developmental neurone (Nedelec etc., 1992; Shirasawa etc., 1993; Rougon etc., 1991) and in embryo intestinal cells, nose cell, salivary gland cell, kidney of rats epithelial cell (hirasawa etc., 1993), reproducibility muscle (Figarella-Branger etc., 1993).CD24 disappears usually from the cell that reaches its final differential period.The expression of CD24 is subject to induced strong and is contained (Allman etc., 1992 once again at the ripening period of T cell and B cell subsequently; Bruce etc., 1981; Crispe and Bevan, 1987; Hardy etc., 1991; Husmann etc., 1988; Linton etc., 1989; Symington and Hakamori, 1984; Takei etc., 1981).Red corpuscle is an exception, because it has maintained high-caliber CD24, expresses.CD24 also is expressed in keratinocyte (Magnaldo and Barrandon, 1996), epidermal langerhan cell (epidermal Langerhans cell) (Enk and Katz, 1994) and dendritic cell (Inaba etc., 1992; Ardavin and Shortman, 1992) in.CD24 is expressed in (Jackson etc. 1992) in small cell lung cancer as major surface antigen.It is expressed in (Karran etc., 1995 in kinds cancer; Akashi etc., 1994; Weber etc., 1995).

Nielsen etc. (1997) have produced and have lacked the knock-out mice that CD24 expresses.But these mouse are characterised in that the normal development of T cell and medullary cell demonstrate the blocking-up of seepage in the B cell development, and it has pre B cell and the reduction of immature B cell mass in late period in marrow.Periphery B cell quantity is normal, and does not measure the damaged of immunologic function with panimmunity and infection model inspection in these mouse.Red corpuscle from these mouse demonstrates higher gathering tendency, more responsive for external hypotonic cracking, and has shorter volume lifetime.

Lu etc. (2000) have reported that slight cross of CD24 in transgenic mice express the exhaustion that causes B lymphoidocyte in marrow, its may due to the apoptosis of pre B cell caused necrocytosis increase cause.

Transmembrane protein 211 (TMEM211) genes encoding transmembrane protein.MRNA has four kinds of splice variants, and it comprises following gene and protein sequence:

protein sequence (SEQ ID NO1)

1?mllggwllla?fnaifllswa?vapkglcprr?ssvpmpgvqa?vaatamivgl?lifpiglasp

61?fikevceass?myyggkcrlg?wgymtailna?vlasllpiis?wphttkvqgr?tiifssater

121?iifvpemnk

cDNA sequence (SEQ ID NO.2)

1?ctttgcctgg?aaggtctcag?ctgtgatgct?cctcggaggc?tggctcctgt?tggccttcaa

61?tgcaattttc?ctcctgtctt?gggctgtggc?ccccaaaggg?ctgtgcccaa?ggagaagcag

121?tgttccaatg?ccaggggtgc?aggcagtggc?agctactgcc?atgattgtgg?gtctgctgat

181?tttcccaatc?ggccttgcct?ccccattcat?caaggaagtg?tgcgaagcct?cctccatgta

241?ttatggtggg?aagtgccggc?tgggttgggg?ttacatgact?gctatcctca?atgcagtcct

301?ggccagcctc?ctgcccatca?tcagctggcc?ccacacaacc?aaggtccaag?ggaggaccat

361?catcttctcc?agtgccaccg?agagaatcat?ctttgtgcca?gaaatgaaca?aataaaaatc

421?tcctgggagt?agcacaaagg?gcacactcca?gagttttatg?aaatcatcat?gtagccaact

481?tcaaatccca?tctctgctcc?ttcttgc

TMEM is quite conservative between various different plant species.Promoter Analysis for transcription factor binding site point shows that the motif for CdxA albumen enriches.Homology frame PROTEIN C DX-1 is by the protein of CDX1 genes encoding in the mankind.This gene is the member of the relevant homeobox transcription factor gene family of tail side.Coded DBP regulation and control intestines expression of specific gene and intestinal epithelial cells differentiation.It has demonstrated the expression of inducing intestinal alkaline phosphatase gene, and suppresses beta-catenin/T cytokine transcriptional activity.

The 147 routine samples altogether that use is comprised of the normal persons of 58 example, the 30 example persons of obscuring and 59 routine colorectal carcinomas are implemented the colorectum that the antibody capture by vesica carries out and are detected (Figure 109 C).Obscure sample and comprise the sample with the situation shown in table 48.

Table 48: vesica CRC test obscure sample

The person of obscuring	Sample number
		Rheumatoid arthritis	3
Type ii diabetes, transitional cell carcinoma of bladder	2
		Rheumatoid arthritis, significant degenerative osteoarthritis	2
The Clear cell renal cell carcinomas of diabetes, kidney	1
		Diabetes, wetting property breast ductal cancer	1
Diabetes, renal cell carcinoma	1
		Chronic diverticulitis	9
Lung cancer	11

The ROC of biomarker TMEM211 and CD24 analyzes and is described in respectively in Figure 109 A and Figure 109 B.In the analysis of carrying out with CD24, sensitivity is 92%, and specificity is 90%.Use TMEM211 as capture antibody and use and detect antibody CD9, CD63, CD81, having obtained data in table 49 at above-mentioned sample, detecting CRC:

Table 49: use the CRC that TMEM211 carries out to detect

True positives	59
		True negative	58
False positive	11
		False negative	0
Amount to	128

Sensitivity	Specificity

100.00％

84.06％

In the authenticity follow-up study, TMEM211 is in 225 patients' cohort, detecting colorectal carcinoma, and described cohort comprises that 76 routine CRC samples, 80 routine normal controls and 69 examples obscure sample.Anti-TMEM211 is used as capture antibody, and detection antibody comprises anti-CD9, anti-CD 63 and anti-CD81.Describedly obscure sample shown in table 50.

Table 50: vesica CRC test obscure sample

The person of obscuring	Sample number
		Rheumatoid arthritis	5
Type ii diabetes, transitional cell carcinoma of bladder	3
		The Clear cell renal cell carcinomas of diabetes, kidney	2
The wetting property breast ductal cancer	1
		Chronic diverticulitis	9
Lung cancer	49

The results are shown in Figure 109 D-E of follow-up study.Figure 109 D shows the average fluorescent strength (MFI) of sample while using TMEM211.Use the threshold value (horizontal bar) indicate and the results are shown in table 51 of acquisition.

Table 51: use TMEM211 to detect the result of CRC

True positives	73
		True negative	124
False positive	25
		False negative	3
Gross sample	225
		Sensitivity	96％
Specificity	83％

The ROC of the same sample of assessing with TMEM211 analyzes and is described in Figure 109 E.AUC is 0.952.

Patient's cohort that use is comprised of the plasma sample from normal control, I phase CRC patient, II phase CRC patient, III phase CRC patient and the person of obscuring has been implemented other conclusive evidence Journal of Sex Research.According to described herein, obscure sample from suffering from cancer outside CRC and the patient of Other diseases, comprise the sample from the patient of the infitrating ductal carcinoma of suffering from rheumatoid arthritis, type ii diabetes and transitional cell carcinoma of bladder, diabetes and kidney Clear cell renal cell carcinomas, diabetes and mammary gland, chronic diverticulitis and lung cancer.The blood plasma microcapsule bubble is analyzed performance for detection of CRC shown in table 52.

Table 52:TMEM211 and CD24 detect the performance of CRC

All sample is used the result of TMEM211 and CD24 to illustrate in Figure 109 F.By using the combination of TMEM211 and CD24, described Test Identification 13 examples (100% sensitivity) in 13 routine I phase CRC cancers, 22 examples (96% sensitivity) in 23 routine II phase CRC cancers and 37 examples (93% sensitivity) in 40 routine III phase CRC.TMEM211 and CD24 are separately for distinguishing various different classes of results respectively shown in Figure 109 G and 109H.

embodiment 54: the microRNA in colorectal cancer cell system is crossed expression

TaqMan low density array (TLDA) microRNA card is used for to the relatively miRNA expression of CRC clone to normal vesica.Use is from Applied Biosystems, Foster City, the TaqMan of CA

microRNA is analyzed and array system Collection and analysis miRNA.The Megaplex provided according to manufacturers ^tMpools Quick Reference Card scheme is used the TaqMan of Applied Biosystems

people's microRNA array.Referring to embodiment 17.

Figure 110 describes colorectal carcinoma (CRC) clone in detail and compares with the TLDA miRNA card of normal vesica.Described clone comprises LOVO, HT29, SW260, COLO205, HCT116 and RKO.The figure illustrates described CRC clone and compare 2-3 expression increase doubly with normal control.These microRNAs are not crossed and are expressed in melanoma cell.

The sequence of analyzing in Figure 110 comprises miR-548c-5p, miR-362-3p, miR-422a, miR-597, miR-429, miR-200a and miR-200b.The sequence of microRNA is shown in table 53.

Table 53: microRNA

embodiment 55: for detection of the microRNA of CRC

MicroRNA (miR) is available from from 12 CRC patients and the vesica separated 4 control patients.Analyzed described sample for being accredited as the two kinds of miR (miR 92 and miR 491) that cross expression in CRC clone.Figure 111 A shows, in CRC patient's sample with respect to the normal person also higher level of visible these miR.Figure 111 B shows, the improvement that miR 92 has obtained normally together with miR 491 and the CRC sample is distinguished.Figure 111 C shows and can carry out the interpolation of the other miR of multiple analysis with miR 92 and miR 491.Described figure has shown miR 92, miR 21, miR 9 and miR 491 multiple analysis for detection of CRC.

embodiment 56: the KRAS order-checking in CRC clone and patient's sample

KRAS RNA is separated and is checked order from the vesica from CRC clone.Before order-checking, RNA is transformed into to cDNA.On clone in listing in table 54, checked order:

Table 54:CRC clone and KRAS sequence

Table 54 and Figure 112 show, are detected in the RNA that detected sudden change also comprises at the vesica from described clone in the genomic dna from described clone.Figure 112 shows the cDNA sequence (Figure 112 A) that is derived from vesica mRNA in HCT 116 cells and the sequence (Figure 112 B) of genomic dna.

12 routine CRC patient's samples check order for KRAS.According to shown in table 55, it is all wild-type (WT).During RNA extracts, all patients sample has all been accepted the processing of DNA enzyme.Extract RNA from the vesica separated.Whole 12 patients for the GAPDH amplification show that RNA is present in its vesica.

Table 55:CRC patient's sample and KRAS sequence

In finding patient's sample that the patient suddenlys change positive for KRAS 13G>A, from the KRAS sudden change of CRC patient tumors sample is also can be in being derived from same patient's source plasma vesica identified, go out.The genomic dna (Figure 112 D) that Figure 112 shows the sequence (Figure 112 C) that is derived from the cDNA of vesica mRNA in blood plasma in this patient and is derived from fresh frozen paraffin embedding (FFPE) tumor sample.

embodiment 57: the CRC miR in the vesica part

In the present embodiment, miRNA size in serum found in the vesica part for 100-1000nm (large vesica) for 50-100nm (vesicles) and size compares.

Use has separated RNA from the Exomir test kit of Bioo Scientific (Austin, Texas) from three parts of 1ml colorectal carcinomas (CRC) patient serum sample.This method has been used strainer to separate described large vesica and vesicles part.Before separation, described serum carries out centrifugal to remove cell debris in whizzer.

By the RNA of 40ng, add in the RT-PCR reactant and by Exiqon miRCURY LNA ^tMuniversal RT microRNA PCR Human Panels I and II (Exiqon, Inc, Woburn, MA) are for assessment of the relative expression of 742 kinds of miR.Result is carried out normalization method and used GeneSpring GX 11.0 (Agilent Technologies, Inc., Santa Clara, CA) to be analyzed according to the scheme of manufacturers.Carry out normalization method with the observed value that between plate, calibration and RT-PCR calibrate each sample, use the larger vesica of paired t-test-vesicles matched samples.Not correction p value with 0.05 is determined significance,statistical.It is found that 5 kinds of miR are for differential expression significantly.Referring to table 56.

Table 56: separate from matching the large vesica of sample and the miR level of vesicles colony

The miR detection agent	The p value	The adjusting of the large relative vesicles of vesica	Multiple changes
				hsa-miR-376c	0.0067	Raise	56.4
hsa-miR-652	0.0015	Raise	287.8
				hsa-miR-221 ^*	0.049	Lower	42.1
hsa-miR-215	0.019	Lower	46
				hsa-miR-324-5p	0.028	Raise	36.2

The Identification that multiple changes goes out 361 kinds at the miR that has greatly the difference that surpasses 2 times between vesica and vesicles.There are 8 kinds of miR detect but do not detect in vesicles in large vesica, and have 3 kinds of miR only to detect in vesicles but do not detect in large vesica.Referring to table 57.

Table 57: separate from matching the large vesica of sample and the miR level in vesicles colony

Only in large vesica, detect	Only in vesicles, detect
		hsa-miR-376c	hsa-miR-215
hsa-miR-652	hsa-miR-582-5p
		hsa-miR-324-5p	hsa-miR-1296
hsa-miR-28-5p
		hsa-miR-190
hsa-miR-590-5p
		hsa-miR-202
hsa-miR-195

These data show, in sample, the miRNA content of large vesica and vesicles is similarly, but are not inevitable identical.These differences can be used for the Optimized Diagnosis test.

the combination of embodiment 58:CRC mark

In the present embodiment, according to above being analyzed described in embodiment 52.Sample sets comprises 462 routine samples, and 256 examples are normal (for these purposes are defined as the individuality of not suffering from CRC) from the individual and 206 routine samples of suffering from the CRC confirmed through tissue biopsy.Described 256 routine cancer samples comprise 12 examples not sample, 57 routine I phases, 103 routine II phases, 78 routine III phases and 6 routine IV phases by stages.Normal specimens is the not ill individuality of oneself's statement of the range of age coupling.For the antibodies of marked vesica surface antigen MUC1, GPCR 110, TMEM211 and CD24 on pearl and for catching the microcapsule bubble of plasma sample.CD9, the CD63 of use PE mark, the microcapsule bubble that the CD81 marker beads is caught and mensuration are in conjunction with the fluorescence of vesica.The fluorescence intensity of mark is for classifying sample as CRC with normal.

In Human Gene Ontology (HUGO) database, GPCR 110 also is called as the title g protein coupled receptor 110 (G protein-coupled receptor 110) of checking and approving or the code name GPR110 checked and approved.Referring to www.genenames.org/data/hgnc_data.php? hgnc_id=18990.Two kinds of optional transcripts are confirmed as REFSEQ protein N P_079324.2 and NP_722582.2.Described GPR110 genes encoding cell membrane protein.

The HUGO of MUC1 checks and approves name and is called mucin 1, the cell surface combination.Referring to www.genenames.org/data/hgnc_data.php? hgnc_id=7508.7 kinds of optional transcripts are confirmed as REFSEQ protein N P_001018016.1, NP_001018017.1, NP_001037855.1, NP_001037856.1, NP_001037857.1, NP_001037858.1 and NP_002447.4.The MUC1 gene is member and its membrane-bound glycosylation phosphoric acid albumen of encoding of Saliva Orthana family, and described albumen plays a role in cell adhesion.

MUC1, GPCR 110, TMEM211 and CD24 as capture antibody for detection of the microcapsule bubble in CRC and normal plasma.Use as described the vesica of CD9, CD63 and CD81 mark capturing.Determine that meta fluorescence intensity (MFI) cutoff threshold is to carry out optimal separation by cancer patients and normal person.If the mark of sample improves, to be considered to for colorectal carcinoma (CRC) be positive to this sample.The diagnosis performance of each independent mark is shown in table 58.

Table 58: for microcapsule bubble MFI in the relatively normal plasma sample of single mark CRC

	Muc1	GPCR?110	TMEM211	CD24
					True positives	232	225	235	212
True negative	162	168	151	182
					False positive	44	38	55	26
False negative	24	31	21	46
					Amount to	462

					Sensitivity	90.63％	87.89％	91.80％	82.17％
Specificity	78.64％	81.55％	73.30％	87.50％
					Accuracy	85.28％	85.06％	83.55％	84.55％
The MFI cutoff	390	360	200	600

Table 59-61 shows and uses the detection of various mark combinations to CRC.In table 61, if in logical separation the mark of (that is, "or") arbitrary be positive, think that sample is positive for colorectal carcinoma (CRC).For example, if Muc1 is the positive and GPCR (being GPR110) be positive and TMEM (being TMEM211) or CD24 arbitrary be positive, think " Muc1 & GPCR & (TMEM or CD24) " be positive.By the result by table 58 and the result of table 59-61, compare, observing the unique identification thing can provide the height to CRC and normal specimens to separate accurately, but uses in some cases multiple markers to improve the detection to CRC.

Table 59: for two kinds of relatively normal plasma sample microcapsule bubble MFI of mark combination CRC

Table 60: for the relatively normal plasma sample microcapsule bubble MFI of multiple markers combination CRC

Table 61: for the relatively normal plasma sample microcapsule bubble MFI of multiple markers combination CRC

Figure 113 show wherein TMEM211 and MUC1 as capture antibody the figure for detection of the microcapsule bubble in CRC and normal plasma.As the described vesica that uses CD9, CD63 and CD81 mark capturing.Measure meta fluorescence intensity (MFI) cutoff threshold so that cancer patients and normal person are carried out to optimal separation.If sample improves in two kinds of marks, to be considered to for colorectal carcinoma (CRC) be positive to this sample.The diagnosis performance of each independent mark and TMEM211 and MUC1 combination is above shown in table 58 and 59.

embodiment 59: the biological marking of the vesica of mammary cancer (BCa)

To be connected with pearl for the antibody of a large amount of target antigens and use it for the vesica of catching from 10 experimenters that suffer from mammary cancer or 10 normal persons' (that is, without mammary cancer) blood sample according to the method for embodiment 49-50.Capture antibody is for the described vesica antigen of the present embodiment.Use detects for the fluorescent-labeled antibody of described four transmembrane protein CD9, CD63 and CD81 the vesica that pearl catches.Use laser detection to measure the also meta fluorescence intensity (MFI) of the vesica of mark of catch.

Use capture antibody group to implement described analysis.When use is implemented to analyze for the following capture antibody of following vesica antigen, there is significant difference in the MFI detected between mammary cancer and normal plasma: CD9, HSP70, Gal3, MIS, EGFR, ER, ICB3, CD63, B7H4, MUC1, DLL4, CD81, ERB3, VEGF, BCA225, BRCA, CA125, CD174, CD24, ERB2, NGAL, GPR30, CYFRA21, CD31, cMET, MUC2 and ERB4.

Use 10 routine mammary cancer samples and 10 routine normal specimens and other capture antibody to implement subsequent experimental.Figure 114 A has shown the figure of sign biomarker expressed in description mammary cancer over normal person's multiple variation.Described mark comprises CD9, EphA2, EGFR, B7H3, PSMA, PCSA, CD63, STEAP, STEAP, CD81, B7H3, STEAP1, ICAM1 (CD54), PSMA, A33, DR3, CD66e, MFG-8e, EphA2, Hepsin, TMEM211, EphA2, TROP-2, EGFR, mammary gland globin, Hepsin, NPGP/NPFF2, PSCA, 5T4, NGAL, NK-2, EpCam, NGAL, NK-1R, PSMA, 5T4, PAI-1 and CD45 from left to right.A plurality of posts for same antigen have shown to use the different capture antibodies that may identify different epi-positions.

Figure 114 B shows the level of the various biomarkers that detect in the vesica from breast cancer cell MCF7, T47D and MDA.T47D and MDA are metastatic cell systems.The antigen of observing in breast cancer cell line comprises CD9, MIS Rii, ER, CD63, MUC1, HER3, STAT3, VEGFA, BCA, CA125, CD24, EPCAM and ERB B4.

The ability of analyzing multiple vesica biomarker in single multiple experiment is with for setting up the biological marking, with for mammary cancer with find the best target organisms mark for other biological marking.Identical technology can be applicable to different setting (as, various disease, various cancers, different target organisms mark, diagnosis, prognosis or treatment diagnosis etc.) to identify that the new bio mark is for analysis exploitation subsequently.

embodiment 60: mammary cancer (BCa) the vesica analysis of using multiple analysis to carry out

In the present embodiment, according to the overall process analysis of describing in embodiment 27 from suffering from mammary cancer (BCa) or not suffering from the patient's of BCa (normally) plasma sample.Prepare blood plasma and carry out multiple analysis according to embodiment 50 according to the scheme of embodiment 49.Capture antibody for the vesica surface antigen protein in table 62 is screened for the biomarker to detecting BCa.

Table 62:BCa capture antibody

According to shown in table 62, according to operability and according to required testing needle the Multiple Antibodies to single target organisms mark.This allows to use different surfaces epi-position assessment vesica to catch, to determine any desired properties that is provided for detecting BCa.

embodiment 61: the source plasma vesica in mammary cancer

The circulation microcapsule bubble plays a significant role in several bioprocesss, comprises vascularization and immunomodulatory.The present embodiment is conceived to the level of the specific vesica subgroup that changes in the patient who suffers from disease (especially mammary cancer).Monitoring to the microcapsule bubble subgroup should contribute to the important biomolecule process that discriminating is relevant to the progress of cancer and Other diseases.

For identifying the relevant microcapsule bubble of cancer, compared the microcapsule bubble in patient's sample between the cohort of the normal control relatively do not suffered from breast cancer the patient who suffers from advanced breast cancer.Microcapsule bubble in plasma sample from described cohort (MV) is concentrated, with the antibody of fluorescence dye coupling, dyeed and use flow cytometry analyzed.Tumour-specific, leukocyte specific with the specific antibody of stroma cell for the identification of these hypotypes with characterizing microcapsule bubble in plasma sample.These tissue-specific antibody and process Specific marker (as the DLL4 for the vascularization microcapsule bubble and VEGFR2, for the CTLA4 of immunosuppression microcapsule bubble and FasL and for CD80 and the CD83 of immunostimulation microcapsule bubble) are matched.According to described herein, use for the described vesica of capture antibody mark of the mark of described mark and detect vesica, use subsequently flow cytometry to detect the vesica be labeled.

Compared the circulation microcapsule bubble between patient with breast cancer and normal healthy controls.By surveying that the antigen relevant from vesica is identified and quantitative remarkable different and subgroup that information is provided.Immunosuppression microcapsule bubble (the CD45 of 68% couple 44% that raises in the cancer patients ⁺mV, its coexpression CTLA4).In addition, vascularization MV raises in cancer patients's blood plasma, and it has 44% coexpression DLL4 and the circulation microcapsule bubble of CD31, and this forms contrast with the vesica that shows these marks that has 2% in normal control.These results show, with the blood plasma of patient with advanced cancer, compare, and from the blood plasma of normal individual, comprise the relevant microcapsule bubble of vascularization, inhibitive ability of immunity and cancer that reduces quantity.The circulation microcapsule bubble can provide simple and reliable tools with the important information that obtains the pernicious and cancer process about occurring in the patient without the pathological evaluation of tissue biopsy or standard, such as immunohistochemistry.

embodiment 62: the biological marking of the vesica of lung cancer (LCa)

Use the described method of embodiment 49-50, to be connected with pearl for the antibody of plurality of target antigen and use it for and catch from the experimenter who suffers from lung cancer, normal control (that is, without lung cancer) or suffer from the vesica in experimenter's the plasma sample of Other diseases.Capture antibody is for the described vesica antigen of the present embodiment.Use detects for the fluorescent-labeled antibody of four transmembrane protein CD9, CD63 and CD81 the vesica that pearl catches.Use laser detection to measure the meta fluorescence intensity (MFI) of the vesica of described mark of catching.

As shown as table 63, detected vesica according to the method described above in the plasma sample from 10 routine normal control samples, 10 routine non-lung cancer cancer samples and 10 routine lung cancer samples in first group of experiment.

Table 63: sample

Use is caught the vesica in described sample for the antibody of listing in Figure 115 A and antigen in Figure 115 B with the capture antibody be connected with pearl.Antigen in described figure is from left to right: SPB, SPC, TFF3, PGP9.5, CD9, MS4A1, NDUFB7, Cal3, iC3b, CD63, MUC1, TGM2, CD81, B7H3, DR3, MACC1, TrkB, TIMP1, GPCR (GPR110), MMP9, MMP7, TMEM211, TWEAK, CDADC1, UNC93, APC, A33, CD66e, TIMP1, CD24, ErbB2, CD10, BDNF, ferritin, ferritin, Seprase, NGAL, EpCam, ErbB2, osteopontin (OPN), LDH, OPN, HSP70, OPN, OPN, OPN, OPN, MUC2, NCAM, CXCL12, haptoglobin (HAP), CRP and Gro-α.In the situation that different capture antibodies repeatedly appears having used in same antigen (as Erbb2, ferritin and osteopontin).Described different antibody can be identified the different epi-positions of identical biomarker.

Use detects the vesica of catching for the fluorescent-labeled antibody of CD9, CD63 and CD81.The meta fluorescence level (MFI) detected illustrates on the Y-axis of Figure 115 B.The Fluorescence Ratio of normal specimens and lung cancer sample or the Fluorescence Ratio of normal specimens and non-lung cancer sample have been shown in Figure 115 A.Figure 115 C shows the MFI from the EPHA2 in the sample of patients with lung cancer and normal control (i), CD24 (ii), EGFR (iii) and CEA (iv).

According to embodiment 49-50, gather from the concentrated microcapsule bubble plasma sample of lung cancer and normal patient and analyzed.With 69 patients of 31 kinds of capture antibody screenings for the vesica surface antigen.Figure 115 D shows the average fluorescent strength on Y-axis (MFI) for lung cancer and normal specimens, and capture antibody marks along X-axis.Use detects the vesica of catching for the fluorescent-labeled antibody of CD9, CD63 and CD81.Antigen in described figure is from left to right: SPB, SPC, NSE, PGP9.5, CD9, P2RX7, NDUFB7, NSE, Gal3, osteopontin, CHI3L1, EGFR, B7H3, iC3b, MUC1, mesothelin, SPA, TPA, PCSA, CD63, AQP5, DLL4, CD81, DR3, PSMA, GPCR 110 (GPR110), EPHA2, CEACAM, PTP, CABYR, TMEM211, ADAM28, UNC93a, A33, CD24, CD10, NGAL, EpCam, MUC17, TROP2 and MUC2.The antigen that can distinguish the most lung cancer and normal specimens comprises SPB, SPC, PSP9.5, NDUFB7, Gal3, iC3b, MUC1, GPCR 110, CABYR and MUC17.

In another group related experiment, assessed the independence of vesica surface antigen but the level of overlapping panel has been arranged in 115 routine lung cancer and 78 routine normal specimens.In described lung cancer sample, there are 35 routine I phases, 53 routine II phases and 27 routine III phase lung cancer.As mentioned above, use and catch vesica for the capture antibody of marked surface antigen in the plasma sample from cohort, and use the vesica of catching for the marker detection antibody test of CD9, CD63 and CD81.Result is shown in table 64 and Figure 115 E.Antigen in Figure 115 E is from left to right: CD9, CD63, CD81, B7H3, PRO GRP, CYTO 18, FTH1, TGM2, CENPH, annexin I, annexin V, ERB2, EGFR, CRP, VEGF, CYTO 19, CCL2, osteopontin (OST19), osteopontin (OST22), BTUB, CD45, TIMP, NACC1, MMP9, BRCA1, P27, NSE, M2PK, HCG, MUC1, CEA, CEACAM, CYTO 7, EPCAM, MS4A1, MUC1, MUC2, PGP9, SPA, SPA, SPD, P53, GPCR (GPR110), SFTPC, UNCR2, NSE, INGA3, INTG b4, MMP1, PNT, RACK1, NAP2, HLA, BMP2, PTH1R, PAN ADH, NCAM, CD151, CKS1, FSHR, HIF, KRAS, LAMP2, SNAIL, TRIM29, TSPAN1, TWIST1, ASPH and AURKB.Table 64 is according to the accuracy of distinguishing cancer and non-cancer sample and to described mark classification.Shown in this table, due to reasons such as sample qualities, be not that all marks are used whole samples.

Table 64: the result of lung cancer marker panel

The ability of analyzing multiple vesica biomarker in single multiplication experiment can be used for setting up the biological marking, and it is for lung cancer and find the best target organisms mark for other biological marking.Identical technology can be applicable to different situation (as, various disease, various cancers, different target organisms mark, diagnosis, prognosis or treatment diagnosis etc.) to identify that the new bio mark is for analysis exploitation subsequently.

embodiment 63: as the biological marking of the circulation microcapsule bubble of the instrument of detection of lung cancer

Circulation microcapsule bubble (cMV) is the membrane-bound structure of cell derived, and it exists in a large number in blood.Tumour cell produces a large amount of cMV, and has shown that it produces and the aggressive of tumour and relevant to the resistance for the treatment of.In the present embodiment, analyzed the protein composition of cMV in the patient who suffers from nonsmall-cell lung cancer (NSCLC).By using decision tree, developed the biological marking that measurable tumour exists.

The cMV that will separate from the blood that derives from the patient who suffers from NSCLC exists with the biomarker that compares to confirm to indicate cancer from the cMV in the similar sample of control patients.By using method as herein described, the existence of the antibody of fluorescent mark pearl for detection of cMV in described sample will be coupled to.

By using multiple analysis and decision tree, we have optimized threshold signal to the optimum level that can isolate two colonies.Use the panel of 63 species specificity biomarkers in the initial cohort of 111 patients that suffer from NSCLC and contrast, we have developed the analytical procedure with high specific and sensitivity.The algorithm of this analysis based on derived from decision tree, it comprises four kinds of capture antibodies that use for following vesica biomarker: a kind of general cMV mark (CD81) and three kinds of lung cancer markers (tensio-active agent protein D (SPD), tensio-active agent albumin A (SP-A) and osteopontin (0PN)).Capture antibody by being coupled to pearl according to use described herein and use the detection antibody of anti-four transmembrane proteins and the detection that the detector based on laser carries out is measured the intensity of the fluorescence that comes from the cMV of being combined with marker beads in the blood sample of the cohort of the patient who suffers from the early stage of lung cancer from 40 and 25 contrasts of not suffering from lung cancer.Average fluorescent strength (MFI) value by the algorithm process gained is to distinguish cancer.The tree derivation of described algorithm has been shown in Figure 116.The MFI threshold value of this step of numeral between each mark, it can be beneficial to sensitivity or specificity through adjustment as described.For SPD, it is positive that the MFI higher than 917 is represented as for cancer.If for the MFI of SPD≤917, next step considers the MFI of CD81.For CD81, it is positive that the MFI lower than 627 is represented as for cancer.If for CD81, MFI >=627, next step considers the MFI of SP-A.For SP-A, it is negative that the MFI lower than 375 is represented as for cancer.If for SP-A, MFI >=375, next step considers the MFI of OPN.For OPN, it is positive that the MFI lower than 80 is represented as for cancer.For OPN, it is negative that MFI >=80 are represented as for cancer.

The result that the decision tree of using Figure 116 is analyzed has been shown in table 65.Shown in this table, described analysis is identified the lung cancer positive with 93% sensitivity, 92% specificity and 92% accuracy.

Table 65: the circulation microcapsule bubble of lung cancer detects

embodiment 64: the circulating vesica compared with circulating tumor cell

Circulating tumor cell (CTC) has been used to the disease process of monitoring in the patient who suffers from dissimilar metastatic cancer.But only 50% metastatic breast cancer, 57% metastatic prostate cancer and 18% transitivity colorectal carcinoma blood sample have enough CTC levels of analyzing for clinical labororatory.The level of vesica can be relevant to tumor progression.

Method: by ultracentrifugation, separate the vesica from 1ml blood plasma.The vesica level that CD81 antibody is used for catching and measuring to mammary cancer sample (n=14) and normal healthy controls (n=4).Use Cell Search CTC testing scheme to whole sample measurement CTC.Subsequently, catch the positive vesica of EpCam from metastatic breast cancer (n=10), prostate cancer (n=2) and colorectal carcinoma (n=3) sample and compare with normal healthy controls (n=7).Extract RNA and by qRT-PCT, the expression of microRNA-21 (miR-21) carried out quantitatively from the positive vesica of these EpCam.

Result: 11 examples (78.6%) in 14 routine samples have the CD-81 specificity vesica level (p=0.002) that is significantly higher than visible horizon in 4 routine healthy sample.Referring to Figure 117 A.In the 14 routine samples of analyzing only 7 examples (50%) have and surpass 5 CTC (it is the clinical threshold value for metastatic breast cancer).3 routine cancer samples have the vesica level lower than the CD-81 measurement of normal specimens mean value, wherein have>5 CTC of an example.MiR-21 to the other metastatic cancer sample of 15 example analyzes (wherein have>5 CTC of 5 examples) and finds miR-21 average out to 4.2 * 10 in mammary cancer, prostate cancer and colorectal carcinoma sample respectively ⁶individual, 4.82 * 10 ⁶individual and 5.05 * 10 ⁶individual copy.Referring to Figure 117 B.In contrast, be collected in the average out to of the plasma sample from healthy donors 1.8 * 10 in the EDTA pipe ⁴individual miR21 copy.Referring to Figure 117 B.

Conclusion: the vesica analysis of plasma sample provides monitoring and has followed the trail of the possibility of disease, and it is better than the CTC analysis in some cases.The vesica in tumour source provides the ability that characterizes tumour with original miR composition, and it has proved the possibility that the biomarker based on the tumour-specific vesica of other blood sample is analyzed.

embodiment 65: vesica is from the depletion of blood plasma

Method for cancer diagnostics based on blood must be filtered to select to have for the specified disease type of certain organs the only a few biomolecules of information from the biomolecules of enormous amount.This has produced many challenges, especially when only having a small amount of cellular material to discharge in blood flow.A strategy that overcomes this obstacle is selected and seeks to understand the particular system of body to circulating vesica.Vesica comprises the corpusculum that double-layer of lipoid is sealed, such as apoptotic body, vesicle, exosome, microcapsule bubble and other biological entities as herein described.Referring to table 2 and relevant discussion.Microcapsule bubble provides abundant information source and secreted by most cell types.The circulation microcapsule bubble in endotheliocyte and white corpuscle source is the main circulation microcapsule bubble existed in blood.The present embodiment has utilized the depletion of these more common circulation microcapsule bubbles to allow the more rare microcapsule bubble subgroup of concentration and analysis.

Use method as herein described with CD31 and CD45 coupling magnetic bead.Described pearl is derived to endotheliocyte and leukocytic circulation microcapsule bubble in hatching with this sample of depletion together with human plasma from the patient with breast cancer.According to method as herein described, use simultaneously for 20 kinds not the capture antibody of synantigen utilize the multiple platform based on immune to characterize remaining microcapsule bubble.Referring to embodiment 49-50.After depletion, some highly associated endothelial cell marker things (as DLL4) are consumed significantly together with CD31, and more common microcapsule bubble mark (as CD9) has significant colony to retain.Referring to Figure 118.These data show, can be from patient's sample the special group of depletion vesica.By using magnetic bead for CD45 with depletion vesica from patient's sample, observed similar tendency.

embodiment 66: as the tissue factor of vesica cancer markers

Tissue factor is the protein that blood coagulation is relevant, has been noted that its expression is associated with cancer.Exist multiple bioprocess and tumour to occur relevant with cancer progression (it is expressed and closely be connected with TF).These processes comprise vascularization, cancer cells intrusion, immunity is evaded survives with circulating tumor cell.Be covered on cancer cell because TF expresses the fibrinogen grumeleuse formed, thereby be coated with for these cells provide protectiveness.Known circulation TF improves in cancer patients's serum.Pathologic fibrosis event (such as thromboembolism and apoplexy) is the major cause that in the patient, cancer dependency circulation microcapsule bubble (cMV) dead and expression TF exists.

Figure 119 describes in the vesica from normal (non-cancer) plasma samples of 10 example, 8 routine mammary cancer (BCa) plasma samples and 2 routine prostate cancer (PCa) plasma samples the detection to tissue factor (TF) in detail.According to described herein, use the anti-tissue factor antibodies that is incorporated into microballoon to catch the vesica in plasma sample.Basic skills is referring to embodiment 49-50.Use detects described vesica of catching for the traget antibody of four transmembrane protein CD9, CD63 and CD81.The figure shows by the viewed meta fluorescence intensity of laser detection (MFI).The MFI of described BCa and PCa sample is as one man higher than normal specimens.In various cancer, detecting of tissue factor shown to TF can be used as cancer vesica mark.

embodiment 67: for cancer, select candidate therapeutic

Can use method of the present invention to identify the biological marking that is used for the treatment of diagnosing mammary cancer.The described biological marking can comprise multiple available biomarker, and it can be assessed according to described herein.The described biological marking can determine in humoral sample, and blood sample preferably, such as blood plasma or serum.The method of using this paper to provide obtains vesica from the humoral sample of the patient from suffering from cancer.For example,, referring to the group method of embodiment 49-50.Determine the biological marking for arranging for difference, can be according to discovery mentioned above for being included in the suitable biological marking of the described biological marking.For example, referring to, the biological marking is found part.Can use the wedding agent that is incorporated into microballoon (described in embodiment 48-50) to separate, catch and/or assess described vesica for surface antigen.Can use array or the FACS in embodiment 26 in embodiment 35-36 to separate, catch and/or assess described vesica for surface antigen.Also can use immuno analytical method to catch vesica.Can analyze as required described separated/biomarker useful load in the vesica of catching.Can use laser measuring technology to assess the size of described vesica.

The described biological marking can further comprise other biomarker, such as microRNA.MicroRNA can directly be assessed or can at first be separated from vesica colony by body fluid.For example, referring to, embodiment 12 (obtaining serum); Embodiment 13 (from the serum and plasma isolation of RNA); Embodiment 16 (from vesica, extracting microRNA).Can use RT-PCR (referring to embodiment 14-15) and/or use array analysis (referring to embodiment 17) to assess described microRNA.Can use the microfluid method to implement nucleic acid amplification to analyze described microRNA.

Can in single analysis, implement the method for the identification of organism marking.For example, can use multiple method to assess multiple biomarker.In addition, some in described biomarker can be assessed in single analysis and one or more other biomarkers are assessed in different analyses, and described different analysis is multiple analysis also.For example, multiple vesica surface biological mark can be in the first multiple analysis, assessed, and multiple microRNA can be in the second multiple analysis, assessed.Can be by the result of described the first and second multiple analysis in conjunction with comprise described vesica surface biological mark and the biological marking described microRNA with evaluation.

The described biological marking can comprise useful biomarker arbitrarily, it includes but not limited to the biomarker provided in the context of this paper about various diseases and imbalance, includes but not limited in

embodiment

8,11,28-42,45-47 and 51 mark for prostate cancer; In embodiment 9 and 52-58 for the mark of colorectal carcinoma; In embodiment 59-61 in the mark of mammary cancer and embodiment 62-63 for the mark of lung cancer.

embodiment 68: the associated target of medicine

Be tested and appraised the biological marking that comprises the relevant target of medicine and treat diagnosing cancer.The advantage of this method is can be in the situation that do not consider the susceptibility of the definite described cancer of cancer origin for candidate therapy.On the contrary, the molecular spectra of described tumour self provides the guidance that therapeutical agent is selected.Antibody or fit group are for assessment of ABCC1 in vesica colony, ABCG2, ACE2, ADA, ADH1C, ADH4, AGT, AR, AREG, ASNS, BCL2, BCRP, BDCA1, β III tubulin, BIRC5, B-RAF, BRCA1, BRCA2, CA2, caveolin, CD20, CD25, CD33, CD52, CDA, CDKN2A, CDKN1A, CDKN1B, CDK2, CDW52, CES2, CK 14, CK 17, CK 5/6, c-KIT, c-Met, c-Myc, COX-2, cyclin D1, DCK, DHFR, DNMT1, DNMT3A, DNMT3B, CAM 120/80, ECGF1, EGFR, EML4-ALK merges, EPHA2, epiregulin, ER, ERBR2, ERCC1, ERCC3, EREG, ESR1, FLT1, folacin receptor, FOLR1, FOLR2, FSHB, FSHPRH1, FSHR, FYN, GART, GNRH1, GNRHR1, GSTP1, HCK, HDAC1, hENT-1, Her2/Neu, HGF, HIF1A, HIG1, HSP90, HSP90AA1, HSPCA, IGF-1R, IGFRBP, IGFRBP3, IGFRBP4, IGFRBP5, IL13RA1, IL2RA, KDR, Ki67, KIT, K-RAS, LCK, LTB, lymphotoxin-beta-receptor, LYN, MET, MGMT, MLH1, MMR, MRP1, MS4A1, MSH2, MSH5, Myc, NFKB1, NFKB2, NFKBIA, ODC1, OGFR, p16, p21, p27, p53, p95, PARP-1, PDGFC, PDGFR, PDGFRA, PDGFRB, PGP, PGR, PI3K, POLA, POLA1, PPARG, PPARGC1, PR, PTEN, PTGS2, RAF1, RARA, RRM1, RRM2, RRM2B, RXRB, RXRG, SIK2, SPARC, SRC, SSTR1, SSTR2, SSTR3, SSTR4, SSTR5, Survivin, TK1, TLE3, TNF, TOP1, TOP2A, TOP2B, TS, TXN, TXNRD1, TYMS, VDR, VEGF, VEGEA, VEGFC, VHL, YES1, the existence of ZAP70 and level.Described antibody or fit microballoon in embodiment 48-50 or the array in embodiment 35-363 of being incorporated into.Known these marks play a role in the effect of various chemotherapeutics antagonism proliferative disease.Therefore, can assess described mark to be independent of cancer origin or the type selecting candidate therapeutic for described cancer, although the treatment doctor can take in any other relevant information when selecting described candidate therapeutic, for example, patient's medical history, treatment before this, other test result, cancer feature (as, phase, origin), doctor's experience, etc.

By the existence of each mark and level with do not suffer from described cancer with reference to sample sets in existence and the level of viewed identical mark compare.Identify in patient's sample with described and compared and express and express not enough biomarker with reference to sample.Use medicine-target correlation rule to identify known being effective in and resisted the list that the candidate therapeutic agent of expressing described biomarker or it being expressed to not enough cancer forms, as the U.S. Patent application 12/658,770 that is shown in the table 9-11 of this paper and submits on February 12nd, 2010; International PCT patent application PCT/US2010/000407 that on February 11st, 2010 submits to; International PCT patent application PCT/US2010/54366 that on October 27th, 2010 submits to; And among the U.S. Provisional Patent Application 61/427,788 of submission on December 28th, 2010; All application is incorporated to this paper in full by reference.For example,, referring to " Table 4:Rules Summary for Treatment Selection " in PCT/US2010/54366.Provide the expression level that comprises assessed biomarker and the report of medicine indication list to described treatment doctor.Described doctor uses described report to assist the selection to candidate therapeutic.

embodiment 69: the curative effect of monitoring prostate cancer

Method for detection of the biological marking of vesica of prostate cancer has been described in embodiment 29-32.Detect vesica in the blood sample from the patient.Determine the described biological marking by detecting the existence of following vesica surface antigen in described sample:

A. general vesica (MV) mark: CD9, CD81 and CD63

B. prostate gland MV mark: PCSA, PSMA

C. relevant MV mark: B7H3, optionally EpCam of cancer

The described biological marking is the treatment effect for prostate cancer for monitoring.The patient is accredited as and has suspicious Serum PSA level (as, blood-serum P SA>4.0ng/ml) and/or suspicious digital rectal examination (DRE).Described patient has been identified to the biological marking of vesica and described result it is found that for prostate cancer to be positive.The treatment doctor determines whether use therapeutical agent, hormonotherapy or operation (prostatectomy) to treat described prostate cancer.After treatment, once again described patient is determined to the biological marking of described vesica.This treatment for the treatment of need to be other for negative patient's reaction and the demonstration of to(for) the positive findings indication of cancer.The negative findings indication is reacted and shows that other treatment may be also nonessential for the positive patient of this treatment.

Also can use the substituting biological marking of prostate cancer, those that include but not limited to provide in

embodiment

8,11,28-42,45-47 or 51.Similarly method is for monitoring the treatment effect to Other diseases and imbalance.For example, can use the biological marking monitoring colorectal carcinoma treatment be described in embodiment 9 or 52-58, use is described in the biological marking monitoring breast cancer treatment in embodiment 59-61, and uses the biological marking monitoring lung cancer therapy be described in embodiment 62-63.These cancers that this paper provides and the biological marking of other disease and imbalance also can be used for monitor therapy effect.

embodiment 70:TMEM211 epitope mapping

Use rabbit polyclonal antibody to survey the TMEM211 peptide library, to identify the epi-position of the TMEM211 be identified on the vesica surface.Long 15 amino acid of peptide and have 12 amino acid whose overlapping.Replace halfcystine with serine residue.All peptides are at the N-terminal biotinylation.Peptide sequence is shown in table 66.

Table 66:TMEM211 overlapping peptide library

Peptide sequence	SEQ?ID?NO.	Peptide sequence	SEQ?ID?NO.
				MLLGGWLLLAFNAIF	10	FIKEVSEASSMYYGG	30
GGWLLLAFNAIFLLS	11	EVSEASSMYYGGKSR	31
				LLLAFNAIFLLSWAV	12	EASSMYYGGKSRLGW	32
AFNAIFLLSWAVAPK	13	SMYYGGKSRLGWGYM	33
				AIFLLSWAVAPKGLS	14	YGGKSRLGWGYMTAI	34
LLSWAVAPKGLSPRR	15	KSRLGWGYMTAILNA	35
				WAVAPKGLSPRRSSV	16	LGWGYMTAILNAVLA	36
APKGLSPRRSSVPMP	17	GYMTAILNAVLASLL	37
				GLSPRRS?SVPMPGVQ	18	TAILNAVLASLLPII	38
PRRSSVPMPGVQAVA	19	LNAVLASLLPIISWP	39
				SSVPMPGVQAVAATA	20	VLASLLPIISWPHTT	40
PMPGVQAVAATAMIV	21	SLLPIISWPHTTKVQ	41
				GVQAVAATAMIVGLL	22	PIISWPHTTKVQGRT	42
AVAATAMIVGLLIFP	23	SWPHTTKVQGRTIIF	43
				ATAMIVGLLIFPIGL	24	HTTKVQGRTIIFSSA	44
MIVGLLIFPIGLASP	25	KVQGRTIIFSSATER	45
				GLLIFPIGLASPFIK	26	GRTIIFSSATERIIF	46
IFPIGLASPFIKEVS	27	IIFSSATERIIFVPE	47
				IGLASPFIKEVSEAS	28	SSATERIIFVPEMNK	48
ASPFIKEVSEASSMY	29

Catch biotinylated peptide in the hole applied at the streptavidin of 96 hole droplet degree plates.Described plate also comprises the hole of the known control peptide of being identified by control antibodies.Use the anti-TMEM211 rabbit polyclonal antibody of one-level of 0.3 μ g/ml or hatch described plate with the rabbit polyclonal control antibodies of 0.3 μ g/ml.Wash described plate and it is hatched together with secondary antibody, described secondary antibody is comprised of the goat anti-rabbit igg that is coupled to horseradish peroxidase (HRP).Use standard method known in the art to carry out the zymetology colorimetric reaction to detect the secondary antibody of combination.After reaction terminating, read each hole of described droplet degree plate with spectrophotometer under the 450nm wavelength.Carry out repeated experiments three times.Positive reading shows that the anti-TMEM211 rabbit polyclonal antibody of described one-level or control antibodies (in the situation that applicable) are incorporated on fixing peptide.

Figure 120 A-C shows the result of described experiment.In described figure, 1-12 (front row, position, from left to right) corresponding to SEQ ID NO:10-21, position 13-24 (front several the 2nd rows, from left to right) corresponding to SEQ ID NO:22-33, (front several the 3rd rows, (arrange, from left to right) corresponding to SEQ ID NO:46-48 corresponding to SEQ ID NO:34-45 and position 37-39 position 25-36 from left to right)

afterwards.Position

40 and 41 is that positive control and position 42 and 43 are negative controls.

Figure 120 A shows the result that described plate and described anti-TMEM211 rabbit polyclonal antibody and goat anti-rabbit igg HRP secondary antibody are hatched.In described figure, the peptide at 30-33 place, position has provided signal clearly.These positions are corresponding to SEQ ID NO:39-42.Figure 120 B shows described plate and contrasts with described the result that rabbit polyclonal antibody and goat anti-rabbit igg HRP secondary antibody are hatched.Positive control has provided strong signal.Figure 120 C shows the result that described plate and described anti-TMEM211 rabbit polyclonal antibody and goat anti-mouse IgG HRP secondary antibody are hatched.Due to described secondary antibody identification mouse IgG, so this experiment also in contrast.Only positive control has provided signal.

According to these experiments, identified a series of overlapping peptides by described anti-TMEM antibody recognition, it is corresponding to SEQ ID NO:39-42.These peptides are equal to N-terminal sequence " LNAVLASLLPIISWPHTTKVQGRT " (SEQ ID NO:49).According to shown in table 67, the common epitope be contained in described four overlapping peptides is PIISWP (SEQ ID NO:50).This shows to use the Identification of the antibodies vesica of the described epi-position PIISWP of identification (SEQ ID NO:50).

Table 67: anti-TMEM211 common epitope

SEQ?ID?NO.	Peptide sequence
		39	LNAVLASLL PIISWP
40	VLASLL PIISWPHTT
		41	SLL PIISWPHTTKVQ
42	PIISWPHTTKVQGRT

embodiment 71:B7H3 epitope mapping

Except indicating place, followed above the described experimentation of embodiment " TMEM211 epitope mapping " so that be identified for can be in conjunction with the epi-position of the anti-B7H3 antibody that is presented in the B7H3 on vesica.One-level antibody is large mouse-anti B7H3 monoclonal antibody.The B7H3 antigen sequence is contained by having 12 overlapping 15-mer overlapping peptides of residue, thereby has obtained 174 peptides.Peptide sequence is illustrated in Figure 68.

Table 68:B7H3 overlapping peptide library

Figure 121 A-C shows the result of described experiment.In described figure, position 1-12 (left bank, from front to back) corresponding to SEQ ID NO:51-62, position 13-24 (the 2nd arranges from left to right, from front to back) corresponding to SEQ ID NO:63-74, position 25-36 (the 3rd arranges from left to right, from front to back) corresponding to SEQ ID NO:75-86, position 37-48 (the 4th arranges from left to right, from front to back) corresponding to SEQ ID NO:87-98, position 49-60 (the 5th arranges from left to right, from front to back) corresponding to SEQ ID NO:99-110, position 61-72 (the 6th arranges from left to right, from front to back) corresponding to SEQ ID NO:112-122, position 73-84 (the 7th arranges from left to right, from front to back) corresponding to SEQ ID NO:123-134, position 85-96 (the 8th arranges from left to right, from front to back) corresponding to SEQ ID NO:135-146, position 97-108 (the 9th arranges from left to right, from front to back) corresponding to SEQ ID NO:147-158, position 109-120 (the 10th arranges from left to right, from front to back) corresponding to SEQ ID NO:159-170, position 121-132 (the 11st arranges from left to right, from front to back) corresponding to SEQ ID NO:171-182, position 133-144 (the 12nd arranges from left to right, from front to back) corresponding to SEQ ID NO:183-194, position 145-156 (the 13rd arranges from left to right, from front to back) corresponding to SEQ ID NO:195-206, position 157-168 (the 14th arranges from left to right, corresponding to SEQ ID NO:207-218 and position 169-174, (the 15th arrange from left to right from front to back), from front to back) corresponding to SEQ ID NO:219-224.Sequence is referring to table 68.Position 175 and 176 is that positive control and position 177 and 178 are negative controls.

Figure 121 A shows the result that plate and anti-B7H3 rat monoclonal antibody and goat Chinese People's Anti-Japanese Military and Political College mouse IgG HRP secondary antibody are hatched.In described figure, in position, the peptide at 10,11,83 and 84 places has provided signal clearly.These positions correspond respectively to SEQ ID NO:60,61,133 and 134.Figure 121 B shows the result that plate and control rats polyclonal antibody and goat Chinese People's Anti-Japanese Military and Political College mouse IgG HRP secondary antibody are hatched.Described positive control has provided strong signal.Figure 121 C shows the result that plate and anti-B7H3 rat monoclonal antibody and goat anti-rabbit igg HRP secondary antibody are hatched.Due to described secondary antibody identification rabbit igg, so this experiment also in contrast.Only positive control has provided signal.

According to these experiments, determined a series of overlapping peptides by anti-B7H3 antibody recognition, it is corresponding to SEQ ID NO:60,61,133 and 134.These peptides are equal to sequence " ALEVQVPEDPVVALVGTDA " (SEQ ID NO:225).According to shown in table 69, the common epitope be contained in described four overlapping peptides is VQVPEDPVVALVG (SEQ ID NO:226).This shows to use the identification Identification of the antibodies vesica of described epi-position VQVPEDPVVALVG (SEQ ID NO:226) all or in part.

Table 69: anti-B7H3 common epitope

SEQ?ID?NO.	Peptide sequence
		60	ALE VQVPEDPVVALV
61	VQVPEDPVVALVGTD
		133	VE VQVPEDPVVALVG
134	QVPEDPVVALVGTDA

embodiment 72:CD9 epitope mapping

Carry out the epi-position that this studies to identify the anti-CD9 monoclonal antibody of mouse, it can be used for detecting the CD9 that is presented in the vesica surface.Carry out epitope mapping by the elutriation phage display peptide library.A plurality of peptides that are incorporated into specifically this anti-CD9 antibody but are not incorporated into control mice IgG2b antibody have been identified.

The material used comprises following: Ph.D.-12 phage display library test kit, numbering No.E8100S, New England Biolabs (Ipswich, MA); Horseradish peroxidase (HRP)/anti-M13 mono-clonal conjugate, numbering No.27-9421-01, GE Healthcare (Waukesha, WI); The maleic anhydride activation clear bar microplate (Amine-binding of amine combination, Maleic Anhydride Activated Clear Strip MicroPlates), numbering No.15100, Pierce, the branch (Rockford, IL) of Thermo Fisher Scientific; M13 sequencing primer (96) 5 '-CCC TCA TAG TTA GCG TAA CG-3 ' (SEQ ID NO.227).Target antibody is the anti-CD9 monoclonal antibody of mouse, and control antibodies is mouse IgG 2b antibody.At phosphate buffered saline (PBS) (PBS; PH 7.6) in dialysis target antibody and control antibodies to improve at described amine in conjunction with the coating efficiency on flat board.Carried out the elutriation of three-wheel library.Use Ph.D. ^tMphage display library instruction manual 1.0 editions, New England BioLabs; And the Mol Immunol such as Stone, the method for describing in 2007,44:2487-91 is tested.Further details provides hereinafter.

In the 1st takes turns elutriation, with the target antibody (200 μ g/ml) through dialysis in PBS (pH 7.6), under 4 ℃, the microplate hole is applied and spends the night.Wash described plate 5 times with PBS-T (0.05%Tween 20 in PBS), and carry out the sealing of 2 hours with M-TBS (2% skimmed milk in tris buffered water salt (TBS)) under 37 ℃.After carrying out 5 washings with PBS-T, by phage library (10 ¹¹pfu) add in blind hole and at room temperature (RT) on vibrator, hatch 1 hour.Discard unconjugated phage and wash described plate 6 times with PBS-T, and wash 3 times in addition with PBS.Carry out 6 minutes hatching and combining phage particle wash-out enters in the 1.5ml centrifuge tube of M-PBS sealing by the triethylamine (TEA) with 200 μ l 0.1M.1M Tris-HCl (pH7.4) with 100 μ l carries out the neutralization of 10 minutes to described particle subsequently.The phage of wash-out is carried out to titration and amplification in 5 hours with for the next round elutriation.

Take turns in taking turns elutriation with the 3rd and used the poor elutriation strategy (subtractive panning strategy) that subtracts the 2nd.Take turns elutriation for each and replace the sealing damping fluid.At first, apply the microplate hole with control antibodies and target antibody respectively, and the 3%BSA be used in TBS is sealed.Apply in hole and hatch in advance the amplification phage (10 of from the 1st, taking turns elutriation in contrast ¹¹pfu) to subdue the phage that is incorporated into mouse IgG 2b.After under RT, difference being subtracted, remaining phage applies in hole and hatches 1 hour at target.Discard phage, titration and the amplification of unconjugated phage and elution of bound for the 3rd, to take turns elutriation.The 3rd elutriation process of taking turns elutriation is similar to the 2nd and takes turns, and unique difference is to seal damping fluid and replaces with 2% M-TBS.

After the three-wheel elutriation, choose 92 single plaques for phage Enzyme Linked Immunoadsorbent Assay (ELISA), to test and clone likely is checked order.Overnight incubation in e. coli k12 strain ER2738 culture by the phage stoste of 1ml in 100ml LB substratum; The dilution culture of 1ml is allocated in each hole of 96 deep-well plates.92 holes with take turns together with the plaque of titre plate and hatch from the 3rd, and hatch two positive controls (E12, F12) and negative control (G12, H12) in 4 remaining holes.Flat board with 250rpm at 37 ℃ of lower oscillation incubation 4.5-5 hour.Subsequently under 4 ℃ with the centrifugal described plate of 4,000rpm 30 minutes.Collect supernatant liquor for the Phage-ELISA for described target and control antibodies.

Apply two 96 hole microplates by target or the control antibodies with 1 μ g/ml under 4 ℃, wash described plate 3 times with 0.05% PBS-T, under 37 ℃, with 2%M-PBS, seal 2 hours, the repeated washing step, add the collected supernatant liquor of 100 μ l in each hole, hatch subsequently described plate and spend the night under 4 ℃, thereby carry out ELISA.Repeated washing and detect any residual phage by carrying out hatching in 1 hour with the anti-M13 antibody of HRP/ of dilution in 1: 5000 under 37 ℃.Described plate is developed the color and measured absorbancy under 450nm by colorimetric analysis.Identify positive colony according to described absorbancy.The single stranded DNA (ssDNA) that extracts positive bacteriophage is with for sequencing analysis.

Figure 122 A-B shows the result of with target antibody (Figure 122 A) or anti-mouse IgG control antibodies (Figure 122 B), the gained phage from the three-wheel elutriation being carried out to the ELISA screening.In described figure, position 1A-12D is corresponding to the positive colony of above identifying.Position E12 and F12 are positive controls, and it comprises the amplification library of from the described the 2nd and the 3rd, taking turns the gained phage of elutriation.Position H12 and G12 are negative controls, the supernatant liquor that it comprises the ER2738 culture.

The peptide sequence that phage was presented of selecting 20 positive colonies to be identified by described anti-CD9 target antibody with announcement for order-checking.According to shown in table 70, sequencing result has identified several peptides.In table 70, clone's numbering is corresponding to the position in the hole shown in Figure 88 A.

Table 70: by with anti-CD9, carrying out the peptide that the phage library screening is identified

The positive colony numbering	Peptide sequence	Clone's number	SEQ?ID?NO.
				E1、F1、D3、D5、F5、C7、C9	RINMNYMINHMM	7	228
C1、B5、F7、F9、D12	AIGWNYPTEMIR		5					229
				H5、A12	HTAKQMMSYMIR	2	230
H3	WFYDSQMIMDSA		1					231
				A5	SMIHRLQAHNIM	1	232
C5	SMWHTLGRHWIA		1					233
				E5	THRDASWIRADL	1	234
E9	VPLHHSQMYPPK		1					235
				H9	WLHPAQPVNHMF	1	236

Generally speaking, after the elutriation of three-wheel library and Phage-ELISA analysis, find that several positive colonies are incorporated into target antibody.Select 20 kinds of positive colonies to extract and order-checking for ssDNA.Identify 9 kinds of peptide sequences from sequencing result.According to shown in table 70,7 kinds of clones present sequence RINMNYMINHMM (SEQ ID NO.228), separately there are 5 kinds to present sequence A IGWNYPTEMIR (SEQ ID NO.229), and separately have 2 kinds to present sequence HTAKQMMSYMIR (SEQ ID NO.230).All the other 6 kinds clones (SEQ ID NO.231-236) are unique.Described peptide can be used as the epitope mimic thing for resisting the quantitative measurment of CD9 antibody amount.

Although the preferred embodiments of the invention are demonstrated in this article and described, clearly these embodiments are only provided in an exemplary fashion for a person skilled in the art.Those skilled in the art can expect a large amount of transformations, change and replacement without departing from the invention.The multiple different replacement scheme that should understand embodiment of the present invention as herein described can be used to implement the present invention.In therefore method and structure in following claim intention definition scope of the present invention and these claim scopes and their equivalent covered in.

Claims

1. one kind characterizes the method that prostate gland is lacked of proper care, it comprises existence or the level of determining from one or more biomarkers listed in table 5 in experimenter's biological sample or table 9-11, the biological marking of the existence that evaluation comprises described one or more biomarkers or level, and the described biological marking and reference are compared, characterize thus described prostate gland imbalance.

2. method according to claim 1, wherein said prostate gland imbalance comprises that BPH and described one or more biomarkers are selected from BCMA, CEACAM-1, HVEM, IL-1R4, IL-10Rb, Trappin-2, p53, hsa-miR-329, hsa-miR-30a, hsa-miR-335, hsa-miR-152, hsa-miR-151-5p, hsa-miR-200a, hsa-miR-145, hsa-miR-29a, hsa-miR-106b, hsa-miR-595, hsa-miR-142-5p, hsa-miR-99a, hsa-miR-20b, hsa-miR-373, hsa-miR-502-5p, hsa-miR-29b, hsa-miR-142-3p, hsa-miR-663, hsa-miR-423-5p, hsa-miR-15a, hsa-miR-888, hsa-miR-361-3p, hsa-miR-365, hsa-miR-10b, hsa-miR-199a-3p, hsa-miR-181a, hsa-miR-19a, hsa-miR-125b, hsa-miR-760, hsa-miR-7a, hsa-miR-671-5p, hsa-miR-7c, hsa-miR-1979, hsa-miR-103 and combination thereof.

3. method according to claim 1, wherein said prostate gland imbalance comprises that prostate cancer and described one or more biomarkers are selected from CD9, PSMA, PCSA, CD63, CD81, B7H3, IL 6, OPG-13, IL6R, PA2G4, EZH2, RUNX2, SERPINB3, EpCam, hsa-let-7b, hsa-miR-107, hsa-miR-1205, hsa-miR-1270, hsa-miR-130b, hsa-miR-141, hsa-miR-143, hsa-miR-148b ^*, hsa-miR-150, hsa-miR-154 ^*, hsa-miR-181a ^*, hsa-miR-181a-2 ^*, hsa-miR-18a ^*, hsa-miR-19b-1 ^*, hsa-miR-204, hsa-miR-2110, hsa-miR-215, hsa-miR-217, hsa-miR-219-2-3p, hsa-miR-23b ^*, hsa-miR-299-5p, hsa-miR-301a, hsa-miR-301a, hsa-miR-326, hsa-miR-331-3p, hsa-miR-365 ^*, hsa-miR-373 ^*, hsa-miR-424, hsa-miR-424 ^*, hsa-miR-432, hsa-miR-450a, hsa-miR-451, hsa-miR-484, hsa-miR-497, hsa-miR-517 ^*, hsa-miR-517a, hsa-miR-518f, hsa-miR-574-3p, hsa-miR-595, hsa-miR-617, hsa-miR-625 ^*, hsa-miR-628-5p, hsa-miR-629, hsa-miR-634, hsa-miR-769-5p, hsa-miR-93, hsa-miR-96 and combination thereof.

4. method according to claim 1, wherein said prostate gland imbalance comprises that prostate cancer and described one or more biomarkers are selected from A33, a33n15, AFP, ALA, ALIX, ALP, annexin V, APC, ASCA, ASPH (246-260), ASPH (666-680), ASPH (A-10), ASPH (D01P), ASPH (D03), ASPH (G-20), ASPH (H-300), AURKA, AURKB, B7H3, B7H4, BCA-225, BCNP1, BDNF, BRCA, CA125 (MUC16), CA-19-9, C-Bir, CD1.1, CD10, CD174 (Lewis y), CD24, CD44, CD46, CD59 (MEM-43), CD63, CD66e CEA, CD73, CD81, CD9, CDA, CDAC11a2, CEA, C-Erb2, C-erbB2, CRMP-2, CRP, CXCL12, CYFRA21-1, DLL4, DR3, EGFR, Epcam, EphA2, EphA2 (H-77), ER, ErbB4, EZH2, FASL, FRT, FRT c.f23, GDF15, GPCR, GPR30, Gro-α, HAP, HBD 1, HBD2, HER 3 (ErbB3), HSP, HSP70, hVEGFR2, iC3b, IL 6Unc, IL-1B, IL6Unc, IL6R, IL8, IL-8, INSIG-2, KLK2, L1CAM, LAMN, LDH, MACC-1, MAPK4, MART-1, MCP-1, M-CSF, MFG-E8, MIC1, MIF, MIS RII, MMG, MMP26, MMP7, MMP9, MS4A1, MUC1, MUC1 seq1, MUC1 seq11A, MUC17, MUC2, Ncam, NGAL, NPGP/NPFF2, OPG, OPN, p53, p53, PA2G4, PBP, PCSA, PDGFRB, PGP9.5, PIM1, PR (B), PRL, PSA, PSMA, PSME3, PTEN, R5-CD9 Tube 1, Reg IV, RUNX2, SCRN1, seprase, SERPINB3, SPARC, SPB, SPDEF, SRVN, STAT 3, STEAP1, TF (FL-295), TFF3, TGM2, TIMP-1, TIMP1, TIMP2, TMEM211, TMPRSS2, TNF-α, Trail-R2, Trail-R4, TrKB, TROP2, Tsg 101, TWEAK, UNC93A, VEGF A, YPSMA-1 and combination thereof.

5. method according to claim 1, it is metastatic or invasive that wherein said sign comprises described characterizing prostate cancer.

6. method according to claim 5, wherein said one or more biomarkers are selected from hsa-miR-100, hsa-miR-1236, hsa-miR-1296, hsa-miR-141, hsa-miR-146b-5p, hsa-miR-17 ^*, hsa-miR-181a, hsa-miR-200b, hsa-miR-20a ^*, hsa-miR-23a ^*, hsa-miR-331-3p, hsa-miR-375, hsa-miR-452, hsa-miR-572, hsa-miR-574-3p, hsa-miR-577, hsa-miR-582-3p, hsa-miR-937, miR-10a, miR-134, miR-141, miR-200b, miR-30a, miR-32, miR-375, miR-495, miR-564, miR-570, miR-574-3p, miR-885-3p and combination thereof.

7. method according to claim 1, wherein said sign comprises determining whether described experimenter processes and react over against treatment, or described experimenter processes and may respond or reactionless for treatment.

8. method according to claim 7, wherein said treatment is processed and is selected from table 8-10.

9. method according to claim 7, wherein said treatment is processed and is comprised radical prostatectomy and/or radiotherapy.

10. method according to claim 7, wherein said treatment is processed to be selected to observe and is waited for, the surgery pelvic lymphadenectomy, radical prostatectomy, transurethral prostatectomy (TURP), testectomy, radiotherapy, external radiation exposure radiotherapy (EBRT), iodine I 125, palladium, iridium, hormonotherapy, the luteinising hormone-releasing hormo agonist, bright dried meat Li Te, goserelin, buserelin, antiandrogen, flutamide, bicalutamide, Magace, Nilutamide, KETOKONAZOL, aminoglutethimide, gonadotropin releasing hormone (GnRH), oestrogenic hormon, psychrotherapy, chemotherapy, biotherapy, ultrasonic and proton beam radiation.

11. method according to claim 7, wherein hsa-miR-1974, hsa-miR-27b, hsa-miR-103, hsa-miR-146a, hsa-miR-22, hsa-miR-382, hsa-miR-23a, hsa-miR-376c, hsa-miR-335, hsa-miR-142-5p, hsa-miR-221, hsa-miR-142-3p, hsa-miR-151-3p, hsa-miR-21, hsa-miR-16.

12. method according to claim 1, the step wherein described biological marking compared with described reference comprises whether the relative described reference of any biomarker of determining in described one or more biomarkers changes to some extent, and determining that prognosis, diagnosis or the treatment of described prostate gland imbalance are diagnosed is provided thus.

13. method according to claim 1, wherein said one or more biomarkers comprise 5T4, ACTG1, ADAM10, ADAM15, ALDOA, ANXA2, ANXA6, APOA1, ATP1A1, BASP1, Clorf58, C20orfl14, C8B, CAPZA1, CAV1, CD151, CD2AP, CD59, CD9, CD9, CFL1, CFP, CHMP4B, CLTC, COTL1, CTNND1, CTSB, CTSZ, CYCS, DPP4, EEF1A1, EHD1, ENO1, F11R, F2, F5, FAM125A, FNBP1L, FOLH1, GAPDH, GLB1, GPX3, HIST1H1C, HIST1H2AB, HSP90AB1, HSPA1B, HSPA8, IGSF8, ITGB1, ITIH3, JUP, LDHA, LDHB, LUM, LYZ, MFGE8, MGAM, MMP9, MYH2, MYL6B, NME1, NME2, PABPC1, PABPC4, PACSIN2, PCBP2, PDCD6IP, PRDX2, PSA, PSMA, PSMA1, PSMA2, PSMA4, PSMA6, PSMA7, PSMB1, PSMB2, PSMB3, PSMB4, PSMB5, PSMB6, PSMB8, PTGFRN, RPS27A, SDCBP, SERINC5, SH3GL 1, SLC3A2, SMPDL3B, SNX9, TACSTD1, TCN2, THBS1, TPI1, TSG101, TUBB, VDAC2, VPS37B, YWHAG, one or more in YWHAQ and YWHAZ.

14. method according to claim 1, wherein said one or more biomarkers comprise one or more prostate specific biomarkers that are selected from CD9, CD63, CD81, PSMA, PCSA, B7H3 and EpCam.

A 15. method that characterizes benign prostatic hyperplasia (BPH), it comprises existence or the level of determining from one or more biomarkers listed in table 5 in experimenter's biological sample, the biological marking of the existence that evaluation comprises described one or more biomarkers or level, and the described biological marking and reference are compared.

A 16. method that characterizes prostate cancer, it comprises existence or the level of determining from one or more biomarkers listed in table 5 in experimenter's biological sample, the biological marking of the existence that evaluation comprises described one or more biomarkers or level, and the described biological marking and reference are compared.

A 17. invasive method that characterizes prostate cancer, it comprises existence or the level of determining from one or more biomarkers listed in table 5 in experimenter's biological sample, the biological marking of the existence that evaluation comprises described one or more biomarkers or level, and the described biological marking and reference are compared.

18., according to the described method of any one in claim 1 or 15-17, wherein said biological sample comprises body fluid.

19. method according to claim 18, wherein said body fluid comprises peripheral blood, serum, blood plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, marrow, synovia, aqueous humor, amniotic fluid, earwax, milk, bronchoalveolar lavage fluid, seminal fluid, prostatic fluid, examine amber liquid or the liquid of ejaculating in advance, the women penetrates liquid, sweat, movement, hair, tear, capsule liquid, hydrothorax and ascites fluid, pericardial fluid, lymph liquid, the food gruel, chyle, bile, interstitial fluid, menses, fester, sebum, vomitus, vaginal secretions, mucous membrane secretory product, just rare, pancreatic juice, nasal lavage fluid, the broncho-pulmonary aspirated liquid, blastochyle or Cord blood.

20., according to the described method of any one in claim 1 or 15-17, wherein said biological sample comprises urine, blood or blood derivatives.

21., according to the described method of any one in claim 1 or 15-17, wherein said biological sample comprises one or more microcapsule bubbles.

22. method according to claim 21, wherein said one or more biomarkers are associated with described one or more microcapsule bubbles.

23. method according to claim 21, wherein said one or more microcapsule bubbles have the diameter of 20nm to 800nm.

24. method according to claim 21, wherein said one or more microcapsule bubbles have the diameter of 20nm to 200nm.

25. method according to claim 21, wherein said one or more microcapsule bubbles carry out that size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanometer film ultrafiltration, immunosorption are caught, affinity purification, affinity capture, immunoassay, microfluidic separation, flow cytometry or their combination.

26. method according to claim 21, wherein said one or more microcapsule bubbles contact with one or more wedding agents.

27. method according to claim 26, wherein said one or more wedding agents comprise nucleic acid, DNA molecular, RNA molecule, antibody, antibody fragment, fit, class peptide, zDNA, peptide nucleic acid(PNA) (PNA), lock nucleic acid (LNA), lectin, peptide, arborescence, membrane protein labelling agent, chemical compound or its combination.

28. method according to claim 26, wherein said one or more wedding agents are for catching and/or detect described one or more microcapsule bubbles.

29. method according to claim 26, wherein said one or more wedding agents one or more surface antigens on described one or more microcapsule bubbles are combined.

30. method according to claim 29, wherein said one or more surface antigens comprise one or more protein.

31. method according to claim 30, wherein said one or more protein comprise one or more in CD9, CD63, CD81, PSMA, PCSA, B7H3 and EpCam.

32. method according to claim 30, wherein said one or more protein comprise one or more in the protein in four transmembrane proteins, CD9, CD63, CD81, CD63, CD9, CD81, CD82, CD37, CD53, Rab-5b, annexin V, MFG-E8 or table 3.

33. method according to claim 26, wherein said one or more wedding agents are for catching described one or more microcapsule bubbles.

34. method according to claim 33, wherein said one or more biomarkers comprise the useful load in described one or more captive microcapsule bubbles.

35. method according to claim 34, wherein said useful load comprises one or more nucleic acid, peptide, protein, lipid, antigen, carbohydrate and/or proteoglycan.

36. method according to claim 35, wherein said nucleic acid comprises one or more DNA, mRNA, microRNA, snoRNA, snRNA, rRNA, tRNA, siRNA, hnRNA or shRNA.

37. method according to claim 35, wherein said nucleic acid comprises one or more microRNAs that are selected from hsa-miR-200b, hsa-miR-375, hsa-miR-141, hsa-miR-331-3p, hsa-miR-181a, hsa-miR-574-3p and combination thereof.

38. method according to claim 35, wherein said nucleic acid comprises one or more microRNAs that are selected from table 27-41.

A 39. method that characterizes colorectal carcinoma, it comprises existence or the level of determining from one or more biomarkers listed in table 6 in experimenter's biological sample and table 9-11, the biological marking of the existence that evaluation comprises one or more biomarkers described in described biological sample or level, and the described biological marking and reference are compared, characterize thus described colorectal carcinoma.

40., according to the described method of claim 39, wherein said one or more biomarkers are selected from miR-92, miR-21, miR-9, miR-491, hsa-miR-376c, hsa-miR-215, hsa-miR-652, hsa-miR-582-5p, hsa-miR-324-5p, hsa-miR-1296, hsa-miR-28-5p, hsa-miR-190, hsa-miR-590-5p, hsa-miR-202, hsa-miR-195 and combination thereof.

41., according to the described method of claim 39, wherein said one or more biomarkers comprise one or more in Muc1, GPCR 110, TMEM211 and CD24.

42., according to the described method of claim 39, wherein said one or more biomarkers comprise A33, AFP, ALIX, ALX4, ANCA, APC, ASCA, AURKA, AURKB, B7H3, BANK1, BCNP1, BDNF, CA-19-9, CCSA-2, CCSA-3& 4, CD10, CD24, CD44, CD63, CD66CEA, CD66e CEA, CD81, CD9, CDA, C-Erb2, CRMP-2, CRP, CRTN, CXCL12, CYFRA21-1, DcR3, DLL4, DR3, EGFR, Epcam, EphA2, FASL, FRT, GAL3, GDF15, GPCR (GPR110), GPR30, GRO-1, HBD 1, HBD2, HNP1-3, IL-1B, IL8, IMP3, L1CAM, LAMN, MACC-1, MGC20553, MCP-1, M-CSF, MIC1, MIF, MMP7, MMP9, MS4A1, MUC1, MUC17, MUC2, Ncam, NGAL, NNMT, OPN, p53, PCSA, PDGFRB, PRL, PSMA, PSME3, Reg IV, SCRN1, Sept-9, SPARC, SPON2, SPR, SRVN, TFF3, TGM2, TIMP-1, TMEM211, TNF-α, TPA, TPS, Trail-R2, Trail-R4, TrKB, TROP2, Tsg 101, TWEAK, one or more in UNC93A and VEGFA.

43., according to the described method of claim 39, wherein said one or more biomarkers comprise one or more in CD9, EGFR, NGAL, CD81, STEAP, CD24, A33, CD66E, EPHA2, ferritin, GPR30, GPR110, MMP9, OPN, p53, TMEM211, TROP2, TGM2, TIMP, EGFR, DR3, UNC93A, MUC17, EpCAM, MUC1, MUC2, TSG101, CD63, B7H3, CD24 and TETS.

44. according to the described method of claim 39, wherein said sign comprise described colorectal carcinoma is accredited as metastatic or invasive.

45. according to the described method of claim 39, wherein said sign comprises determining whether described experimenter processes and just react for treatment, or described experimenter processes and may respond or reactionless for treatment.

46., according to the described method of claim 45, wherein said treatment is processed and is selected from table 8-10.

47. according to the described method of claim 45, wherein said treatment is processed and is selected from main operative therapy, local excision, the removal of the excision of primary lesion and anastomosis and peripheral lymphoglandula, adjuvant therapy, Fluracil (5-FU), capecitabine, folinic acid, oxaliplatin, Tarceva, irinotecan, acetylsalicylic acid, ametycin, Sutent, Cetuximab, rhuMAb-VEGF, the polyoxyethylene glycol filgrastim, Victibix, draw wooden Xidan anti-, celecoxib, combination treatment, the FOLFOX4 scheme, the FOLFOX6 method, the FOLFIRI scheme, the FUFOX scheme, the FUOX scheme, the IFL scheme, the XELOX scheme, 5-FU and LEVAMISOLE HCL scheme, German AIO scheme, the CAPOX scheme, Douillard scheme and radiotherapy.

48. according to the described method of claim 39, the step wherein described biological marking compared with described reference comprises whether the relative described reference of any biomarker of determining in described one or more biomarkers changes to some extent, and prognosis, the diagnosis to described colorectal carcinoma is provided thus or treats determining of diagnosis.

49., according to the described method of claim 39, wherein said one or more biomarkers comprise A26C1A, A26C1B, A2M, ACAA2, ACE, ACOT7, ACP1, ACTA1, ACTA2, ACTB, ACTBL2, ACTBL3, ACTC1, ACTG1, ACTG2, ACTN1, ACTN2, ACTN4, ACTR3, ADAM10, ADSL, AGR2, AGR3, AGRN, AHCY, AHNAK, AKR1B10, ALB, ALDH16A1, ALDH1A1, ALDOA, ANXA1, ANXA11, ANXA2, ANXA2P2, ANXA4, ANXA5, ANXA6, AP2A1, AP2A2, APOA1, ARF1, ARF3, ARF4, ARF5, ARF6, ARHGDIA, ARPC3, ARPC5L, ARRDC1, ARVCF, ASCC3L1, ASNS, ATP1A1, ATP1A2, ATP1A3, ATP1B1, ATP4A, ATP5A1, ATP5B, ATP5I, ATP5L, ATP5O, ATP6AP2, B2M, BAIAP2, BAIAP2L1, BRI3BP, BSG, BUB3, C1orf58, C5orf32, CAD, CALM1, CALM2, CALM3, CAND1, CANX, CAPZA1, CBR1, CBR3, CCT2, CCT3, CCT4, CCT5, CCT6A, CCT7, CCT8, CD44, CD46, CD55, CD59, CD63, CD81, CD82, CD9, CDC42, CDH1, CDH17, CEACAM5, CFL1, CFL2, CHMP1A, CHMP2A, CHMP4B, CKB, CLDN3, CLDN4, CLDN7, CLIC1, CLIC4, CLSTN1, CLTC, CLTCL1, CLU, COL12A1, COPB1, COPB2, CORO1C, COX4I1, COX5B, CRYZ, CSPG4, CSRP1, CST3, CTNNA1, CTNNB1, CTNND1, CTTN, CYFIP1, DCD, DERA, DIP2A, DIP2B, DIP2C, DMBT1, DPEP1, DPP4, DYNC1H1, EDIL3, EEF1A1, EEF1A2, EEF1AL3, EEF1G, EEF2, EFNB1, EGFR, EHD1, EHD4, EIF3EIP, EIF3I, EIF4A1, EIF4A2, ENO1, ENO2, ENO3, EPHA2, EPHA5, EPHB1, EPHB2, EPHB3, EPHB4, EPPK1, ESD, EZR, F11R, F5, F7, FAM125A, FAM125B, FAM129B, FASLG, FASN, FAT, FCGBP, FER1L3, FKBP1A, FLNA, FLNB, FLOT1, FLOT2, G6PD, GAPDH, GARS, GCN1L1, GDI2, GK, GMDS, GNA13, GNAI2, GNAI3, GNAS, GNB1, GNB2, GNB2L1, GNB3, GNB4, GNG12, GOLGA7, GPA33, GPI, GPRC5A, GSN, GSTP1, H2AFJ, HADHA, hCG_1757335, HEPH, HIST1H2AB, HIST1H2AE, HIST1H2AJ, HIST1H2AK, HIST1H4A, HIST1H4B, HIST1H4C, HIST1H4D, HIST1H4E, HIST1H4F, HIST1H4H, HIST1H4I, HIST1H4J, HIST1H4K, HIST1H4L, HIST2H2AC, HIST2H4A, HIST2H4B, HIST3H2A, HIST4H4, HLA-A, HLA-A29.1, HLA-B, HLA-C, HLA-E, HLA-H, HNRNPA2B1, HNRNPH2, HPCAL1, HRAS, HSD17B4, HSP90AA1, HSP90AA2, HSP90AA4P, HSP90AB1, HSP90AB2P, HSP90AB3P, HSP90B1, HSPA1A, HSPA1B, HSPA1L, HSPA2, HSPA4, HSPA5, HSPA6, HSPA7, HSPA8, HSPA9, HSPD1, HSPE1, HSPG2, HYOU1, IDH1, IFITM1, IFITM2, IFITM3, IGH@, IGHG1, IGHG2, IGHG3, IGHG4, IGHM, IGHV4-31, IGK@, IGKC, IGKV1-5, IGKV2-24, IGKV3-20, IGSF3, IGSF8, IQGAP1, IQGAP2, ITGA2, ITGA3, ITGA6, ITGAV, ITGB1, ITGB4, JUP, KIAA0174, KIAA1199, KPNB1, KRAS, KRT1, KRT10, KRT13, KRT14, KRT15, KRT16, KRT17, KRT18, KRT19, KRT2, KRT20, KRT24, KRT25, KRT27, KRT28, KRT3, KRT4, KRT5, KRT6A, KRT6B, KRT6C, KRT7, KRT75, KRT76, KRT77, KRT79, KRT8, KRT9, LAMA5, LAMP1, LDHA, LDHB, LFNG, LGALS3, LGALS3BP, LGALS4, LIMA1, LIN7A, LIN7C, LOC100128936, LOC100130553, LOC100133382, LOC100133739, LOC284889, LOC388524, LOC388720, LOC442497, LOC653269, LRP4, LRPPRC, LRSAM1, LSR, LYZ, MAN1A1, MAP4K4, MARCKS, MARCKSL1, METRNL, MFGE8, MICA, MIF, MINK1, MITD1, MMP7, MOBKL1A, MSN, MTCH2, MUC13, MYADM, MYH10, MYH11, MYH14, MYH9, MYL6, MYL6B, MYO1C, MYO1D, NARS, NCALD, NCSTN, NEDD4, NEDD4L, NME1, NME2, NOTCH1, NQO1, NRAS, P4HB, PCBP1, PCNA, PCSK9, PDCD6, PDCD6IP, PDIA3, PDXK, PEBP1, PFN1, PGK1, PHB, PHB2, PKM2, PLEC1, PLEKHB2, PLSCR3, PLXNA1, PLXNB2, PPIA, PPIB, PPP2R1A, PRDX1, PRDX2, PRDX3, PRDX5, PRDX6, PRKAR2A, PRKDC, PRSS23, PSMA2, PSMC6, PSMD11, PSMD3, PSME3, PTGFRN, PTPRF, PYGB, QPCT, QSOX1, RAB10, RAB11A, RAB11B, RAB13, RAB14, RAB15, RAB1A, RAB1B, RAB2A, RAB33B, RAB35, RAB43, RAB4B, RAB5A, RAB5B, RAB5C, RAB6A, RAB6B, RAB7A, RAB8A, RAB8B, RAC1, RAC3, RALA, RALB, RAN, RANP1, RAP1A, RAP1B, RAP2A, RAP2B, RAP2C, RDX, REG4, RHOA, RHOC, RHOG, ROCK2, RP11-631M21.2, RPL10A, RPL12, RPL6, RPL8, RPLP0, the RPLP0-sample, RPLP1, RPLP2, RPN1, RPS13, RPS14, RPS15A, RPS16, RPS18, RPS20, RPS21, RPS27A, RPS3, RPS4X, RPS4Y1, RPS4Y2, RPS7, RPS8, RPSA, RPSAP15, RRAS, RRAS2, RUVBL1, RUVBL2, S100A10, S100A11, S100A14, S100A16, S100A6, S100P, SDC1, SDC4, SDCBP, SDCBP2, SERINC1, SERINC5, SERPINA1, SERPINF1, SETD4, SFN, SLC12A2, SLC12A7, SLC16A1, SLC1A5, SLC25A4, SLC25A5, SLC25A6, SLC29A1, SLC2A1, SLC3A2, SLC44A1, SLC7A5, SLC9A3R1, SMPDL3B, SNAP23, SND1, SOD1, SORT1, SPTAN1, SPTBN1, SSBP1, SSR4, TACSTD1, TAGLN2, TBCA, TCEB1, TCP1, TF, TFRC, THBS1, TJP2, TKT, TMED2, TNFSF10, TNIK, TNKS1BP1, TNPO3, TOLLIP, TOMM22, TPI1, TPM1, TRAP1, TSG101, TSPAN1, TSPAN14, TSPAN15, TSPAN6, TSPAN8, TSTA3, TTYH3, TUBA1A, TUBA1B, TUBA1C, TUBA3C, TUBA3D, TUBA3E, TUBA4A, TUBA4B, TUBA8, TUBB, TUBB2A, TUBB2B, TUBB2C, TUBB3, TUBB4, TUBB4Q, TUBB6, TUFM, TXN, UBA1, UBA52, UBB, UBC, UBE2N, UBE2V2, UGDH, UQCRC2, VAMP1, VAMP3, VAMP8, VCP, VIL1, VPS25, VPS28, VPS35, VPS36, VPS37B, VPS37C, WDR1, YWHAB, YWHAE, YWHAG, YWHAH, one or more in YWHAQ and YWHAZ.

50., according to the described method of claim 39, wherein said biological sample comprises body fluid.

51. according to the described method of claim 50, wherein said body fluid comprises peripheral blood, serum, blood plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, marrow, synovia, aqueous humor, amniotic fluid, earwax, milk, bronchoalveolar lavage fluid, seminal fluid, prostatic fluid, examine amber liquid or the liquid of ejaculating in advance, the women penetrates liquid, sweat, movement, hair, tear, capsule liquid, hydrothorax and ascites fluid, pericardial fluid, lymph liquid, the food gruel, chyle, bile, interstitial fluid, menses, fester, sebum, vomitus, vaginal secretions, mucous membrane secretory product, just rare, pancreatic juice, nasal lavage fluid, the broncho-pulmonary aspirated liquid, blastochyle or Cord blood.

52., according to the described method of claim 39, wherein said biological sample comprises ight soil, blood or blood derivatives.

53., according to the described method of claim 39, wherein said biological sample comprises one or more microcapsule bubbles.

54., according to the described method of claim 53, wherein said one or more biomarkers are associated with described one or more microcapsule bubbles.

55., according to the described method of claim 53, wherein said one or more microcapsule bubbles have the diameter of 20nm to 800nm.

56., according to the described method of claim 53, wherein said one or more microcapsule bubbles have the diameter of 20nm to 200nm.

57. according to the described method of claim 53, wherein said one or more microcapsule bubbles carry out that size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanometer film ultrafiltration, immunosorption are caught, affinity purification, affinity capture, immunoassay, microfluidic separation, flow cytometry or their combination.

58., according to the described method of claim 53, wherein said one or more microcapsule bubbles contact with one or more wedding agents.

59., according to the described method of claim 58, wherein said one or more wedding agents comprise nucleic acid, DNA molecular, RNA molecule, antibody, antibody fragment, fit, class peptide, zDNA, peptide nucleic acid(PNA) (PNA), lock nucleic acid (LNA), lectin, peptide, arborescence, membrane protein labelling agent, chemical compound or its combination.

60., according to the described method of claim 58, wherein said one or more wedding agents are for catching and/or detect described one or more microcapsule bubbles.

61., according to the described method of claim 58, wherein said one or more wedding agents one or more surface antigens on described one or more microcapsule bubbles are combined.

62., according to the described method of claim 61, wherein said one or more surface antigens comprise protein.

63., according to the described method of claim 62, wherein said one or more protein comprise one or more in the listed protein of four transmembrane proteins, CD9, CD63, CD81, CD63, CD9, CD81, CD82, CD37, CD53, Rab-5b, annexin V, MFG-E8 or table 3.

64., according to the described method of claim 58, wherein said one or more wedding agents are for catching described one or more microcapsule bubbles.

65., according to the described method of claim 64, wherein said one or more biomarkers comprise the useful load in described one or more captive microcapsule bubbles.

66., according to the described method of claim 65, wherein said useful load comprises one or more nucleic acid, peptide, protein, lipid, antigen, carbohydrate and/or proteoglycan.

67., according to the described method of claim 66, wherein said nucleic acid comprises one or more DNA, mRNA, microRNA, snoRNA, snRNA, rRNA, tRNA, siRNA, hnRNA or shRNA.

68., according to the described method of claim 66, wherein said nucleic acid comprises one or more microRNAs or its combination that is selected from table 6.

69., according to the described method of aforementioned any claim, wherein said method is implemented in vitro.

70. the purposes for the reagent of implementing the described method of aforementioned any claim any one.

71. a test kit, it comprises the reagent that implements the claims the described method of any one in 1-69.

72. the vesica of a separation, it comprises one or more biomarkers that are selected from table 5.

73. the vesica of a separation, it comprises one or more biomarkers that are selected from table 6.

74. the vesica of a separation, it comprises one or more biomarkers that are selected from table 9-11.