CN106018822A

CN106018822A - NY-ESO-1 antigen detection method constructed based on specific monoclonal or polyclonal antibody

Info

Publication number: CN106018822A
Application number: CN201610319103.3A
Authority: CN
Inventors: 朱永良; 杨赛赛; 黄建; 孙红祥
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2016-05-13
Filing date: 2016-05-13
Publication date: 2016-10-12

Abstract

The invention discloses an NY-ESO-1 antigen detection method constructed based on a specific monoclonal or polyclonal antibody. According to the method, NY-ESO-1recombinant protein is used as an antigen for preparing the specific NY-ESO-1 antibody. The NY-ESO-1 antibody and latex microspheres are coupled to prepare antibody conjugate, the conjugate and a peripheral blood sample to be detected are mixed to react 5-10 min, the light absorption value at the 546-nm position is detected, and the NY-ESO-1 antigen concentration in the sample is obtained according to a standard curve. The invention further provides a chemiluminiscence detection method adopting a direct light emitting mode. According to the detection method for the NY-ESO-1 antigen in peripheral blood, automatic large sample detection can be carried out on large automatic instrument such as a biochemical analyzer and a chemiluminiscenceimmunity analyzer. The detection sensitivity of the method is 0.01-10 ng/mL.

Description

The NY-ESO-1 Detection of antigen set up based on specific monoclonal or polyclonal antibody Method

(1) technical field

The present invention relates to NY-ESO-1 antigen detection method in a kind of body fluid, particularly to one based on specific monoclonal Or the detection method of the NY-ESO-1 antigen of polyclonal antibody foundation.

(2) background technology

Cancer-testis antigen (cancer-testis antigen, CTA) is as a kind of important tumor associated antigen, many Plant in tumor cell and express, but be only limitted to testis and embryonal tissue in the normal tissue.CTA has promotion tumor and is formed, opposing Tumor death, promotes the biological functions such as neoplasm metastasis.NY-ESO-1 is the important member in Cancer-testis antigen family, swollen Tumor antigen have more strongly immunogenic, wide expression in kinds of tumors, express the most hardly, the most send out Existing more than 20 CD4⁺Or CD8⁺T cell identification epi-position, and in kinds of tumors, can detect that spontaneous immunoreation.NY-ESO-1 Express and internal spontaneity immunne response and diagnosing tumor, prognosis are relevant, also play an important role at therapeutic field of tumor.Due to life Cell colonization does not express HLA molecule and blood-testis barrier exists, and the immunotherapy of tumors general normal tissue cell for CTA does not produces Raw immunotoxicity, current NY-ESO-1 is considered as the preferable target antigen of immunotherapy of tumors.

NY-ESO-1 antigen first by Chen etc. by recombinant cDNA library serological analysis technology (Serological Analysis of recombinant cDNA expression libraries, SEREX) screen from human esophageal carcinoma Arrive.Hereafter, multinomial research display NY-ESO-1 is multiple in neuroblastoma, synovial sarcoma, malignant melanoma, ovarian cancer etc. Tumor tissues is all expressed higher, colorectal cancer, hepatocarcinoma, bladder transitional cell carcinoma, multiple myeloma, pulmonary carcinoma also have certain journey Degree is expressed.NY-ESO-1 gene family includes tri-members of NY-ESO-1, LAGE-1 and ESO3, is all positioned X chromosome q28, Gene order has certain similarity.It is different from NY-ESO-1 and LAGE-1 specific expressed in tumor tissues, the extensive table of ESO3 Reach in the various normal structure of human body, do not have tumor restricted.NY-ESO-1 gene mRNA about 700bp, the longest up to 747bp, compile The code usual 543bp in district, coded protein relative molecular weight about 18kD, be made up of 180 aminoacid, and its N end has rich in glycine, There is hydrophobic amino acid sequence in C end, has potential trans-membrane region.

NY-ESO-1, as the stronger CTA of immunogenicity, can activate CD4⁺And CD8⁺T lymphocyte, induction body produces pin Cell and humoral immunoresponse(HI) to tumor.In NY-ESO-1 peptide chain, T lymphocyte identification epi-position is mainly with HLA-A2 and HLA- The restricted antigenic peptides of DR is main.The autoimmune response that NY-ESO-1 excites may be suppressed by tumor microenvironment, thus affects Graft Versus Tumor.Therefore, immunotherapy of tumors needs by transformation immunocyte, strengthens immunne response and Graft Versus Tumor is come real Existing.Document report p155-163 (QLSLLMWIT), p157-165 (SLLMWITQC) and p157-167 (SLLMWITQCFL) at present Three antigenic peptides can induce NY-ESO-1 positive tumor patient to produce ctl response, can be applicable to immunotherapy of tumors.

NY-ESO-1, as a kind of important tumor associated antigen, at early diagnosis of tumor, develops, therapeutic effect etc. Aspect has important value.NY-ESO-1 expresses in kinds of tumors tissue, and expresses the most in the normal tissue, and it is swollen Tumor diagnosis has relatively high specific.NY-ESO-1 and autoantibody thereof are combined with routine clinical tumor markers can improve disease The accuracy of diagnosis and sensitivity.Research finds that the routine tumor markerses such as NY-ESO-1 antibody and CEA and CA19-9 combine inspection When surveying gastric cancer, III phase and IV phase patients with gastric cancer accuracy rate of diagnosis improve 11.2%, improve in all patients with gastric cancer 5.8%.Peng etc. are to 112 example Nasopharyngeal Carcinoma Patients and comparison contrast, found that the associating of NY-ESO-1 and VCA-IgA autoantibody Examination can improve sensitivity and the specificity of detection, and Nasopharyngeal Carcinoma Patients is had early by prompting NY-ESO-1 specific autoantibody detection Phase diagnostic effect, can as a potential United screening mark (Peng YH, et al, Oncology letters 2014,8: 1096-1102.).Also having research to have detected 237 example patients with esophageal squamous cells and 134 example matched groups, result shows NY- ESO-1 is one of potential mark of esophageal squamous cell carcinoma early diagnosis (Xu YW, et al.The American journal of gastroenterology2014,109:36-45.).Lai etc. are to 50 example synovial sarcomas, 155 example gastrointestinal stromal tumors, 135 examples Spindle cell sarcoma and the various sarcoma of 77 examples find that NY-ESO-1 presents in synovial sarcoma continually and steadily after carrying out SABC Express, can differentiate in other sarcomas (Lai JP, et al.Oncoimmunology 2012,1:1409-1410.).NY-ESO-1 Judging the most significant at tumor prognosis, research finds that tumor cells expression NY-ESO-1 prompting prognosis is poor.At mammary gland marrow In sample cancer patient, estrogen receptor negative person's tumor tissues NY-ESO-1 positive rate is higher than estrogen receptor positive person, NY-ESO- 1 positive patient is less than NY-ESO-1 negative patient life cycle.Also after having research that G. cephalantha patient is carried out multiplicity Think that NY-ESO-1 is the individual index of its poor prognosis.Additionally, NY-ESO-1 is also one of nonsmall-cell lung cancer prognostic marker (Ademuyiwa FO,et al.PloS one 2012,7:e38783；Laban S,et al.International journal of cancer.2014,135:1142-1152；John T,et al.PloS one 2013,8:e67876.).

In immunotherapy of tumors, CTA advantage is specific cell and body fluid to exempt from selective excitation tumor patient body Epidemic disease is reacted, and damages normal structure hardly.NY-ESO-1 is as having more strongly immunogenic member in CTA, at oncotherapy In play key player.

CTA participates in initial stage fetal development and stem cell self renewal, and it is expressed in tumor tissues and is confined to have The cell of stem cell properties, CT gene expression cell is probably the result of abnormal tumor stem cell clone propagation.Express the swollen of CTA Oncocyte loses differentiation capability, and may participate in tumor recurrence and transfer.In tumor stem cell, CT gene expression is applied In tumor recurrence and the treatment of transfer.There is research to isolate tumor stem cell from glioma cell line and tissue, compare In noble cells, tumor stem cell, NY-ESO-1 expresses notable rising.Additionally, CT gene includes NY-in tumor stem cell The promoter region of ESO-1 shows higher acetylation of histone and methylation level.Due to HLA I class antigenic expression not with Differentiation state changes, and CTA can be as the surface antigen at tumor stem cell, and CT gene such as NY-ESO-1 is proposed to be used in colloid Tumor patient tumors stem cell target vaccine.

The a series of CT antigen associated treatment tumor vaccines having been developed that in recent years, as the NY-ESO-1 with synthetic resists Former peptide vaccine, using cowpox NY-ESO-1 and bird pox NY-ESO-1 antigenic peptides as viral vaccine, all creates controlling in various degree Therapeutic effect.The NY-ESO-1 polypeptide vaccine of the restricted antigenic peptides of HLA-A2 can produce after having been used for clinical trial, and vaccination Raw CD8+T cellullar immunologic response.The research such as Karbach is aided with the NY-ESO-1 peptide of incomplete freund adjuvant and CpG 7909 (p157-165) vaccine immunity HLA-A2 positive late tumor patient, can detect that enduring T cell immunoreation, and this research is thought The NY-ESO-1 peptide vaccine of associating adjuvant is expected to suppress tumor growth (Karbach J, et al.International journal of cancer 2010,126:909-918.).Another research is to NY-ESO-1b peptide and incomplete freund adjuvant Inoculate high-risk epithelial ovarian carcinoma patients safety and immunogenicity is estimated, found that 3 example patients are at 25,38 and 52 There is complete clinical remission in the moon.At home, drug and food Surveillance Authority of China has passed through the evaluation of NY-ESO-1b polypeptide vaccine, It can induce liver cancer patient to produce specificity cellular immunity response, thus effectively suppresses Postoperative Residual tumor cell, is expected to into For one effective tumor aid treatment means.

Adoptive cellular immunotherapy (adoptive cellular immunotherapy, ACT) is special by having antigen The opposite sex identifies that the immunocyte of function feeds back in the patient after amplification in vitro, plays Graft Versus Tumor.Research shows higher ratio The late stage melanomas of example is expressed NY-ESO-1, Rapoport etc. and is expressed patients with malignant myeloma T cell for NY-ESO-1 affinity The φt cell receptor (TCR-T) strengthened, patient, after receiving autologous stem cell transplantation, internal inputs about 2,400,000,000 heredity again Transformation T cell, attacks and expresses NY-ESO-1 tumor cell.Result shows that in 20 patients with terminal, 16 people create notable clinical anti- Should, 14 people (70%) after the treatment in three months close to complete incidence graph or complete incidence graph, the median progression-free survival phase is 19.1 Month, Overall survival is 32.1 months.Patient can generally treat by tolerize T cells, and genetic modification immunocyte has migrated at bone marrow Demonstrate long-term anti-tumor capacity, and tumor recurrence generally (Rapoport AP, et relevant with engineering T cell forfeiture al.Nature medicine 2015,21:914-921.).Robbins etc. are by NY-ESO-1 antigen specific T CR genetic modification T cell 6 synovial cell sarcom of input and 11 malignant melanoma patients, show 4 synovial cell sarcom and 5 pernicious melanocytes Tumor patient has therapeutic effect.Although research display tcr gene modifies T cell preferable clinical efficacy, but still has Partial tumors to suffer from Person remains to escape immunization therapy.After the treatment of NY-ESO-1 specific T-cells, the reason of patients with recurrent generation immune escape may be with Cells in vivo MHC lose relevant (Robbins PF, et al.Journal of clinical oncology 2011,29: 917-924.)。

CART be by identify tumor antigen single-chain antibody scFv (single chain variable fragment, ScFv) combine with T cell activation motif, proceed to T lymphocyte by transduction vector system so that it is there is specific recognition and kill Hinder tumor cell ability.There is research design NY-ESO-1 peptide specific CART cell, and by these improved anti-NY-ESO- 1-T1-CD28/CD3 ζ T cell feeds back to NY-ESO-1 positive mice model, found that a large amount of cytokine secretion, has anti- Tumor effect.

NY-ESO-1 expresses and can control as some early diagnosis of tumor and the important indicator of Index for diagnosis, NY-ESO-1 immunity Treating and also achieve certain progress, NY-ESO-1 is the brightest to cellular biology of tumor activity influence and mechanism of action thereof etc. Really.Immunization therapy based on NY-ESO-1 will provide New Policy for oncotherapy.In detection body fluid especially in peripheral blood Expressing with or without NY-ESO-1, the auxiliary diagnosis and the curative effect that cannot be only used for tumor are dynamically observed it can also be used to whether judge patient It is suitable for the personalized immunization therapy for NY-ESO-1 antigen, and the basis that tumor cell is to immunization therapy effecting reaction.

(3) summary of the invention

It is an object of the present invention to provide a kind of NY-ESO-1 detection side set up based on specific monoclonal or polyclonal antibody Method, including solubility NY-ESO-1 specific monoclonal or the preparation method of polyclonal antibody, and (includes blood for peripheral blood Clearly, blood plasma and whole blood) etc. the detection of NY-ESO-1 proteantigen in body fluid, provide a kind of new swollen for tumor high-risk diagnosis Tumor markers, also the personalized immunization therapy such as precisely treatment and/or CAR-T, TCR-T etc. for NY-ESO-1 positive tumor provides and depends on According to.

The technical solution used in the present invention is: specific binding principle based on antigen Yu antibody, first passes through restructuring egg The anti-NY-ESO-1 monoclonal of white inducing producing specificity or polyclonal antibody, the most again to specific monoclonal or Anti-TNF-α After body is marked, finally detect.

The present invention provides a kind of NY-ESO-1 detection method set up based on specific monoclonal or polyclonal antibody, described Method is: (described anti-NY-ESO-1 antibody is anti-NY-to prepare anti-NY-ESO-1 antibody with NY-ESO-1 recombiant protein for antigen ESO-1 monoclonal IgG antibody (including IgG1, IgG2a, IgG2b, IgG3 or IgG4) or polyclone IgG antibody (comprise F (ab)’₂Specific Ab fragments)), antibody coupling matter is prepared in anti-NY-ESO-1 antibody and latex microsphere coupling；By reagent a with Testing sample mixes, 25-37 DEG C of reaction 3-5min, adds antibody coupling matter, 25-37 DEG C of reaction 5-10min, detects 546nm Place's light absorption value, obtains NY-ESO-1 recombiant protein concentration in testing sample according to standard curve；Described reagent a quality group becomes: 35g/L PEG-4000, bovine serum albumin 5g/L, solvent is the buffer of pH6.8；Described standard curve is by such as lower section Prepared by method: be that antigen immune rabbit prepares anti-NY-ESO-1 specific antibody, by anti-NY-ESO-1 with NY-ESO-1 recombiant protein Specific antibody conjugate is prepared in specific antibody and latex microsphere coupling, and NY-ESO-1 recombiant protein is pressed 0-250ng/ml ladder Degree concentration is diluted to the diluent of 0-250ng/mL with the buffer containing volumetric concentration 0.1-1% bovine serum albumin, with containing institute The buffer stating bovine serum albumin is blank, by the diluent of described variable concentrations and reagent b with mix, 25-37 DEG C Reaction 3-5min, is subsequently adding specific antibody conjugate, 25-37 DEG C of reaction 5-10min, and light absorption value at detection 546nm, to inhale Light value is vertical coordinate, and in diluent, NY-ESO-1 recombiant protein concentration makes standard curve as abscissa；Described reagent b matter Amount composition with reagent a (reagent a of the present invention, reagent b are reagent, different and name for ease of distinguishing different step consumption, Letter itself does not has implication)；Described anti-NY-ESO-1 specific antibody is anti-NY-ESO-1 specific monoclonal IgG antibody or anti- NY-ESO-1 specific polyclonal IgG antibody.

Further, described latex microsphere is with a diameter of 80-400nm, and surface active group is-COOH or-NH₂Quality dense 10% microsphere latex solution form of spending adds.

Further, described antibody coupling matter preparation method is: (1) latex microsphere activates: by latex microsphere and pH6.5, 0.05mol/L MES buffer, water, tween 20 mix, and vortex oscillation mixes；50mg/mL NHS is added water-soluble in mixed liquor Liquid and 10mg/mL carbodiimide aqueous solution, vortex oscillation 15 minutes, more ultrasonic 10 times, the most ultrasonic 5 seconds, it is spaced 30 seconds, ultrasonic Energy 9000kJ；After ultrasonic end, Sephadex G-25 sephadex column crossed by mixed liquor, washes with water to final volume as initially to add Add 2 times of latex microsphere volume, it is thus achieved that the latex solution of activation；Described latex microsphere is with diameter 80-400nm, surface active group For-COOH or-NH₂Mass concentration 10% microsphere latex solution form add；Described latex microsphere with MES buffer volume ratio is 0.4:1, described water and MES buffer volume ratio are 1.5:1, and described tween 20 is that 1-2:100 is (excellent with MES buffer volume ratio Select 2:100)；Described NHS aqueous solution and MES buffer volume ratio are 1:10-20 (preferably 1:16), described carbodiimide aqueous solution It is 1:10-20 (preferably 1:16) with MES buffer volume ratio；(2) coupling: anti-NY-ESO-1 monoclonal or polyclone IgG are resisted Body is dissolved in pH7.4,100mM PBS makes antibody-solutions, latex solution mixing antibody-solutions and step (1) activated After, react 4 hours in vertical mixed instrument；Add pH7.2,1.0mol/L glycine buffer to mix in vertical mixed instrument 10-60 minute, reactant liquor 20000rpm is centrifuged 30 minutes, abandons supernatant, retain precipitation, by the resuspended precipitation of cleaning mixture, 20000rpm is centrifuged 30 minutes, repeats resuspended and centrifugal 3-5 time, takes last centrifugal precipitation and is antibody coupling matter；Described wash Wash liquid and refer to pH7.4,500mM glycine buffer containing volumetric concentration 0.1% bovine serum albumin；The latex of described activation Liquid volumetric usage is calculated as 0.1-1.0ml/mg with anti-NY-ESO-1 monoclonal or polyclonal antibody IgG mass.

Further, described standard curve is prepared as follows: be antigen immune rabbit system with NY-ESO-1 recombiant protein Standby rabbit anti-NY-ESO-1 specific antibody, prepares specific antibody by anti-for rabbit NY-ESO-1 specific antibody and latex microsphere coupling Conjugate, by NY-ESO-1 recombiant protein with the buffer containing volumetric concentration 0.25% bovine serum albumin be diluted to 0ng/mL, The diluent of 5ng/mL, 10ng/mL, 25ng/mL, 50ng/mL, 100ng/mL, 250ng/mL, with containing described bovine serum albumin Buffer be blank, by the NY-ESO-1 recombiant protein diluent of described variable concentrations and reagent b with mix, 25-37 DEG C reaction 5-10min, is subsequently adding specific antibody conjugate, 25-37 DEG C of reaction 5-10min, light absorption value at detection 546nm, With light absorption value as vertical coordinate, in diluent, NY-ESO-1 recombiant protein concentration makes standard curve as abscissa.

Further, described testing sample and reagent a volume ratio are 4:125, described testing sample and antibody coupling matter volume Ratio is 4:25.

Further, the Tris-HCl buffer that solvent is pH6.8,50mM of described reagent preparation a.

Further, the Tris-HCl that buffer is pH6.8,50mM buffering of described dilution anti-NY-ESO-1 specific antibody Liquid.

Additionally, the present invention also provides for a kind of NY-ESO-1 detection side set up based on specific monoclonal or polyclonal antibody Method (i.e. chemoluminescence method), anti-NY-ESO-1 monoclonal that described method is prepared with NY-ESO-1 recombiant protein for antigen or many grams When grand IgG antibody carries out chemiluminescence immunoassay method detection NY-ESO-1 antigen, use direct light-emitting mode, particularly as follows: will be anti- NY-ESO-1 antibody is directly and 2', 6'-dimethyl-carbonyl phenyl 10-methyl-9-acridine formic acid esters 4'-N-succinimide ester three Traget antibody is prepared in methyl fluoride sulfonate (being called for short acridinium ester DMAE-NHS) (CAS:115853-74-2) coupling；By testing sample, Traget antibody, substrate solution A and substrate solution B mixing carries out luminescence-producing reaction, detects reactant liquor luminous intensity, recombinates according to NY-ESO-1 Protein standard curve, it is thus achieved that NY-ESO-1 recombiant protein concentration in testing sample；Described anti-NY-ESO-1 antibody and acridinium ester The ratio of the amount of DMAE-NHS material is 1:3-5；Described anti-NY-ESO-1 antibody is anti-NY-ESO-1 monoclonal IgG antibody or anti- NY-ESO-1 polyclone IgG antibody；Described substrate solution A consists of: mass concentration 0.1-0.25% carbamide peroxide, volumetric concentration 0.01%Triton X-100, solvent is 100mM, pH8.0 Tris buffer, and substrate solution B consists of: 10mM NaOH, volume The Triton X-100 of concentration 2%, solvent is water；Described NY-ESO-1 recombiant protein standard curve preparation method is: NY-ESO- The Tris-HCl buffer of 1 recombiant protein 100mM containing volumetric concentration 0.25% bovine serum albumin, pH7.4 is diluted to 0, The diluent of 5,10,25,50,100,250ng/mL, with the buffer containing described bovine serum albumin of nonantigenic composition as sky White comparison, is separately added into described diluent, adds what anti-NY-ESO-1 antibody was directly prepared with acridinium ester DMAE-NHS coupling Traget antibody, adds substrate solution A and substrate solution B and carries out luminescence-producing reaction, detect reactant liquor luminous intensity, with NY-in diluent ESO-1 recombiant protein concentration is abscissa, and luminous intensity is vertical coordinate, draws standard curve；Described substrate solution A and substrate solution B Form with testing sample detection substrate solution A and substrate solution B used.

Further, described NY-ESO-1 bioassay standard curve is that the luminescence according to NY-ESO-1 recombiant protein gauge orifice is strong Degree, to correct product concentration as abscissa, luminous intensity is vertical coordinate, draws standard curve.

Further, described carbamide peroxide mass concentration is 0.1%.

NY-ESO-1 recombiant protein of the present invention be prepared as approach well known, preferably by PCR method by NY- ESO-1DNA sequence is connected to protokaryon/eukaryotic expression vector, with E.coli DE3 and/or HEK293 or expressing cho cell, Produce and purification NY-ESO-1 recombiant protein.Molecular biology method is used to obtain restructuring from prokaryotic cell and/or eukaryotic cell NY-ESO-1 albumen, particularly carries out renaturation with reduced form-oxidized form of glutathione to NY-ESO-1 with during procaryotic cell expression Rear purification.

Of the present invention prepare specificity mouse monoclonal IgG type (include IgG1 and/or IgG2a and/or IgG2b and/ Or IgG3 and/or IgG4 type) and rabbit source property polyclone IgG antibody (comprise F (ab) '₂Specific Ab fragments) time, thin with protokaryon The restructuring NY-ESO-1 albumen of born of the same parents and/or eukaryotic cell expression carries out immunity acquisition as immunogen.

The present invention is for detecting the former of latex intensified method, ELISA and the chemoluminescence method of NY-ESO-1 antigen in peripheral blood Reason is based on NY-ESO-1 antigen and its specific antibody specific binding.

Compared with prior art, the present invention has the advantages that: latex intensified method of the present invention detection periphery In blood, the method for NY-ESO-1 antigen can be enterprising at the large automatic instrument such as biochemistry analyzer and chemical illumination immunity analysis instrument Row large sample Aulomatizeted Detect.In chemoluminescence method detection peripheral blood, the method for NY-ESO-1 has detection sensitivity height, detection The advantages such as repeatability height and good stability, also can be automated analysis simultaneously.The inventive method detection sensitivity 0.01- 10ng/mL。

(4) accompanying drawing explanation

Fig. 1 is reaction principle schematic diagram, and A is that latex immunoenhancement detects NY-ESO-1 principle schematic, and B is ELISA Method detection NY-ESO-1 principle schematic, C is that chemoluminescence method detects NY-ESO-1 principle schematic；

Fig. 2 is reaction normal curve, and A is that latex immunoenhancement detects NY-ESO-1 standard curve, and B is ELISA method inspection Surveying NY-ESO-1 standard curve, C is that chemoluminescence method detects NY-ESO-1 standard curve.

(5) detailed description of the invention

Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in This:

Embodiment 1

Prepared by 1.NY-ESO-1 recombiant protein

1.1NY-ESO-1DNA sequence obtains

1.1.1 express from NY-ESO-1 and positive tumor cell obtains NY-ESO-1DNA sequence

From tumor cell, NY-ESO-1 positive cell strain is screened by antigen in rinsed fluid method, 1 × 10⁶Express NY-ESO-1 Tumor cell--A375 melanoma cell, with RNAeasy test kit (purchased from Germany Qiagen company) isolated and purified always RNA, through Oligo dT₁₅Total serum IgE reverse transcription is cDNA by primer (purchased from Shanghai Sheng Gong bio-engineering corporation), passes through polymerase chain Formula reaction (PCR) method therefrom expands acquisition NY-ESO-1DNA sequence, and (nucleotides sequence is classified as shown in SEQ ID NO.1, aminoacid Sequence is shown in SEQ ID NO.2), DNA sequencing is verified.

1.1.2 chemosynthesis NY-ESO-1DNA sequence

The NY-ESO-1DNA sequence (protein ID:P78358) provided according to Genbank, entrusts commercial company (nucleotides sequence is classified as shown in SEQ ID NO.1, and aminoacid sequence to learn by SEQ ID NO.2 synthesis NY-ESO-1DNA sequence Show), and be cloned in carrier pUC57.

1.2 prokaryotic cells produce NY-ESO-1 recombiant protein

With step 1.1 obtain NY-ESO-1DNA as template, with polymerase chain reaction (PCR) by NY-ESO-1 DNA Open reading nucleotide sequence (ORF) is cloned into the NdeI/BamHI restriction endonuclease of procaryotic cell expression carrier pET15b or pET14b Site, after DNA sequencing checking, converts Rosetta DE3 escherichia coli, selects the bacterial clone of high expressed NY-ESO-1, expand When cultivating to OD value to 0.4-0.8, inducing 4 hours at 37 DEG C with 1.0mM IPTG, 10000rpm is centrifuged 15 minutes and collects antibacterial Precipitation.

NY-ESO-1 renaturation and purification: illustrate as a example by processing 150g bacterial precipitation.150g bacterial precipitation is added 1.5L inclusion body lysate I (pH8.0,50mM Tris-HCl buffer contains 1mM EDTA and 100mM NaCl) laggard horizontal high voltage Ultrasonication (pressure 10MPa, supersonic frequency 20000Hz, supersound process 5min), centrifugal, precipitation 700ml inclusion body lysate II (pH8.0,50mM Tris-HCl buffer contains 1mM EDTA, 100mM NaCl and 0.5%Triton X-100) washes Washing, wash 2 times, 10000rpm is centrifuged 10min, and the precipitation of generation 0.5M carbamide (uses pH8.0,50mM Tris-HCl buffer Prepare containing 1mM EDTA, 100mM NaCl) wash 1 time, 10000rpm is centrifuged 10min.Precipitation 200ml, 8M carbamide (is used PH8.0,50mM Tris-HCl buffer is prepared containing 1mM EDTA, 100mM NaCl) resuspended, after magnetic agitation 30min, 10000rpm is centrifuged 10min, supernatant (200ml) pH 10.7,50mM K₂HPO₄+ 1mM EDTA+50mM NaCl solution dilutes 10 times, standing 30min, then adjust pH to 8.0 with 2M HCl after adjusting pH10.7 with 1M NaOH, 10000rpm is centrifuged 10min, by upper Clearly (2L) loading bag filter carries out renaturation dialysis.Dialysis solution be pH8.0,50mM Tris-HCl buffer containing 1mM EDTA, 100mM NaCl, 10mM reduced glutathion, 2mM oxidized form of glutathione and 1mM dithiothreitol, DTT (DTT).4 DEG C of renaturation are saturating Analysis 48h, then dialyses with pH8.0,50mM Tris-HCl buffer NaCl Han 100mM, and mistake nickel post after 3 times of dialysing is carried out Purification, eluent is pH8.0,50mM Na₂HPO₄Buffer includes 150mM NaCl and 300mM imidazoles.Pure with AKTA further Changing recombiant protein peak, Lorry ' s phenol reagent carries out quantification of protein.

1.3 eukaryotic cells produce NY-ESO-1 recombiant protein

The NY-ESO-1DNA obtained with step 1.1, as template, is cloned into the eukaryotic expression vector pcDNA3.1 (U.S. Invitrogen company), transfect the engineering cell Chinese hamster ovary celI (U.S. by the method (particle gun or liposome) of physical transfection ATCC company), 5%CO₂In bioreactor, serum-free medium (Gibco company of the U.S.) is cultivated 7-10 days, collects supernatant, Concentrate, with AKTA purification of recombinant proteins peak.Lorry ' s phenol reagent carries out quantification of protein.

1.4NY-ESO-1 recombiant protein is as calibration object or standard substance

The NY-ESO-1 recombiant protein deriving from eukaryotic cell of above-mentioned steps 1.3 preparation, with containing after quantification of protein The 100mM of 0.25%BSA, pH 7.4 phosphate or Tris-HCl buffer be diluted to the recombiant protein concentration containing NY-ESO-1 and be The diluent of 0ng/mL, 5ng/mL, 10ng/mL, 25ng/mL, 50ng/mL, 100ng/mL, 250ng/mL, is adding 0.1% NaN₃Afterwards as calibration object or the standard substance of this detection method.

1.5NY-ESO-1 recombiant protein is as quality-control product

The NY-ESO-1 of the eukaryotic cell expression deriving from prokaryotic cell or step 1.3 preparation of above-mentioned steps 1.2 preparation Recombiant protein, with the 100mM containing 10% human serum or blood plasma after quantification of protein, pH7.4 phosphate or Tris-HCl buffering Liquid is diluted to, containing NY-ESO-1 recombiant protein 5ng/mL, 30ng/mL, 50ng/mL, 150ng/mL liquid not etc., adding 0.1%NaN₃Afterwards as the quality-control product of this detection method.

The most anti-NY-ESO-1 recombiant protein monoclonal IgG type preparation method for antibody

2.1 animal immune

With above-mentioned steps 1.2 or 1.3 preparation NY-ESO-1 recombiant protein, with 100 μ g NY-ESO-1 albumen/time/the least The concentration of Mus mixes with the Split completely of equivalent, subcutaneous multi-point injection immunity Balb/c female mice (5-6 week old, body weight 18-20g), the 1st time with the 2nd minor tick 10-14 days, the most every minor tick 7 days, continuous 5 times, the 2-5 time NY-ESO-1 albumen and The incomplete freund adjuvant emulsifying mixing of equivalent, uses the NY-ESO-1 albumen that tail vein/lumbar injection is aseptic after being spaced 1 week, With booster immunization 1 time, after 3 days, cell merges.

The preparation of 2.2 hybridomies

Carry out according to regular growth fusion method.Take the splenocyte of step 2.1 immune mouse and SP2/0 myeloma cell by Volume ratio 1:6 merges under the effect of 50%PEG (MW400-800), and fused cell is first at hypoxanthine-methotrexate-thymus In pyrimidine (HAT) Selective agar medium, at 37 DEG C, 5%CO₂And cultivate under the conditions of saturated humidity, it is changed to hypoxanthine-breast after 2 weeks Gland pyrimidine (HT) culture medium, continues at 37 DEG C, 5%CO₂And cultivate under the conditions of saturated humidity.In time cloning Kong Changzhi 1/3-1, take Culture supernatant carries out antibody test screening.

2.3 specificity clone's hole sizer choosings

Positive hole is expressed with ELISA method screening antibodies.NY-ESO-1 recombiant protein is diluted with the carbonate buffer solution of 50mM To 10 μ g/mL, it is coated reaction 4 DEG C of overnight or 37 DEG C of 120min of micropore with 100 μ L/ holes, with the phosphorus containing 0.05%Tween-20 Phthalate buffer (PBS) or Tris-HCl buffer (PBS) wash 3 times, close 30min by 10% calf serum room temperature, then add Enter 100 μ l above-mentioned steps 2.2 culture supernatant room temperature reaction 60min, with the phosphate buffer containing 0.05%Tween-20 (PBS) goat anti-mouse igg-HRP room temperature or Tris-HCl buffer (PBS) washs 3 times, finally adding 1:2000 dilution is anti- Answer 60min, wash 3 times with the phosphate buffer (PBS) containing 0.05%Tween-20 or Tris-HCl buffer (PBS), add Enter the colour developing of tetramethyl biphenyl ammonia (TMB) substrate to observe.All there is obvious blue responder for the positive, is otherwise negative.Retain with The cell clone hole that NY-ESO-1 recombiant protein responds, then use limiting dilution assay to carry out 3 time cloning screenings, build the hybridization of strain Oncocyte amplification culture again, frozen, it is used for preparing ascites.

The preparation and purification of 2.4 ascites

After the hybridoma amplification culture of stably excreting monoclonal antibody, with 0.5 × 10⁴～5 × 10⁵Individual/only inoculation To in advance with 0.5mL norphytane or the abdominal cavity of liquid paraffin sensitization 7-10 days Balb/c mice, observe in 7-12 days, collect abdomen Water, measures titer after being centrifuged.Preserve or cryopreservation through 50% saturated ammonium sulphate.Ascites uses 50% saturated ammonium sulfate Precipitation-caprylic acid precipitates, or with the 50% saturated ammonium sulphate-rProtein A-Sepharose affine layer of Fast Flow Analysis purification, finally detects purity with SDS-PAGE, need to reach 90%.Anti-NY-ESO-1 specific monoclonal prepared by this step resists There is specific binding reaction in physical ability and NY-ESO-1 albumen, and not with non-NY-ESO-1 albumen generation nonspecific reaction.

The most anti-NY-ESO-1 recombiant protein polyclone IgG antibody preparation method

With above-mentioned steps 1.2 or 1.3 preparation NY-ESO-1 recombiant protein, with 10mg NY-ESO-1 recombiant protein/time/ Only mix with equivalent Split completely, the immunity of rabbit subcutaneous multi-point injection.Every 1-2 week 1 time, continuous 5 times, adopt after being spaced 1 week With separating carotid artery, collecting blood about 80-120 milliliter, room temperature is placed 2 hours, and 4 DEG C of 10000rpm are centrifuged 30min, separate and collect Serum.Above-mentioned serum is again with NY-ESO-1 recombiant protein-Sepharose 4B affinity chromatograph column purification anti-NY-ESO-1 restructuring egg White specific IgG type antibody, finally detects purity with 8%SDS-PAGE, need to reach 90%.Obtain rabbit source property polyclone IgG Antibody.

For obtaining the F (ab) ' of anti-NY-ESO-1 recombiant protein₂Specific Ab fragments, many grams of the rabbit source property of above-mentioned purification Grand IgG antibody excises IgG's with the pepsin of 0.1% (mass ratio) further in the 50mM acetate buffer of pH 4.5 Fc ' holds, and 37 DEG C are reacted 12 hours, then adjusting or in the case of uncomfortable pH value, with saturated ammonium sulfate and/or SPA and/or NY- ESO-1 affinity chromatograph method purification F (ab) '₂Antibody fragment.Obtain the F (ab) ' of specific anti-NY-ESO-1 recombiant protein₂Special Specific antibody fragment.

Specific binding reaction is there is in anti-NY-ESO-1 Anti-TNF-α physical ability prepared by the present embodiment with NY-ESO-1 albumen, And not with non-NY-ESO-1 albumen generation nonspecific reaction.

4. set up NY-ESO-1 detection method based on specific antibody

4.1 latex immunoenhancements

Latex microsphere crosslinking NY-ESO-1 monoclonal or polyclone IgG antibody method, with rabbit source property polyclone IgG antibody (comprise F (ab) '₂Specific Ab fragments) it is preferential, illustrate as a example by 1% latex-antibody of activation crosslinking 4mL.

4.1.1 latex activation

4.1.1.1 0.05mol/L MES buffer (pH6.5) 1ml, the mass concentration of diameter 80-400nm is 10% glue Breast (Bangs company of the U.S.) 0.4ml, water 2.1mL, 20 μ l tween 20, vortex oscillation mixes.

4.1.1.2 50mg/mL NHS aqueous solution 0.25ml, 10mg/mL carbodiimide (EDAC) aqueous solution 0.25mL are added, Vortex oscillation 15 minutes.

4.1.1.3 ultrasonic 10 times of mixed liquor, the most ultrasonic 5 seconds, are spaced 30 seconds, ultrasonic energy 9000kJ.Cross Sephadex G-25 sephadex column (purchased from Pharmacia), washes with water to final volume 10-20ml, it is thus achieved that the latex solution of activation.

The optimization that the most anti-NY-ESO-1 monoclonal/polyclone IgG antibody cross-links with latex

4.1.2.1 the latex solution 1.0ml of above-mentioned steps 4.1.1 activation, respectively with 0.1,0.2,0.4,0.6,0.8,1.0, The anti-NY-ESO-1 monoclonal/polyclonal antibody of 1.2,1.4,1.6,1.8,2.0mg above-mentioned steps 2.2 preparations (comprises F (ab) '₂ Specific Ab fragments) (being dissolved in respectively in 1.0ml pH7.4,100mM PB buffer) mixing after, hybrid reaction 4 hours.

4.1.2.2 being separately added into 1.0mol/L glycine buffer (pH7.2) 1ml, vertical mixing 60 minutes, to close The active group of residual, it is thus achieved that antibody latex conjugate mixed liquor.

4.1.3 the separation of cross-linking agent

4.1.3.1 the antibody latex conjugate mixed liquor 20000rpm that prepared by above-mentioned steps 4.1.2.2 is centrifuged 30 minutes, abandons Supernatant, retains precipitation.

4.1.3.2 with the resuspended precipitation of cleaning mixture 40ml (cleaning mixture: pH7.4,500mM glycine buffer includes 0.1% BSA), 20000rpm is centrifuged 30 minutes.

4.1.3.3 repeat 4.1.3.2 step 3-5 time, remove free antibody with thorough washing, it is thus achieved that antibody coupling matter.

The most anti-NY-ESO-1IgG monoclonal/polyclone IgG antibody and the preservation of antibody coupling matter

The latex precipitation that above-mentioned steps 4.1.3.3 obtains adds 4ml containing 0.25%BSA, 0.3%PVP, 0.05%NaN₃ PH7.4,100mM glycine buffer, the ultrasonic 5-15 second, ultrasonic energy 9000kJ, 4 DEG C of preservations.

The most anti-NY-ESO-1 monoclonal/polyclone IgG antibody cross-links the determination of optium concentration with latex

4.1.5.1 main agents constituent

Reagent R1: trishydroxymethylaminomethane (Tris-HCl) buffer 50mM, pH6.8；PEG(MW 4000-8000) 35g/L；BSA 5g/L.

Different crosslinking NY-ESO-1 monoclonals or the antibody of polyclonal antibody prepared by reagent R2: above-mentioned steps 4.1.3 are even Connection thing.

4.1.5.2 detecting step

Response type: performance rate method temperature: 37 DEG C of cuvette optical path: 0.6cm

Master/slave wavelength: 546nm/800nm unit: U/ml the Direction of Reaction: rise

4.1.5.3 result determines:

Calculate the difference (A calibration-A is blank) of calibration object absorbance, set up suitable mathematical modulo (non-linear) such as Logit- Log etc., fit to the calibration curve of multiple spot calibration.Reactions change amplitude according to each latex calibration curve, the range of linearity, relevant Coefficient and reaction repeatability determine optimal antibody and latex ratio.

4.1.6 peripheral blood NY-ESO-1 detects

4.1.6.1 main agents constituent

Crosslinking NY-ESO-1 monoclonal prepared by reagent R2: above-mentioned steps 4.1.3 or the antibody coupling matter of polyclonal antibody.

4.1.6.2 detecting step

Response type: 2 method temperature of set time: 37 DEG C of cuvette optical path: 1.0cm

Master/slave wavelength: 546nm/800nm unit: U/ml the Direction of Reaction: rise

4.1.6.3 result calculates:

Calculate the difference (A calibration-A is blank) of calibration object absorbance, set up suitable mathematical modulo (non-linear) such as Logit- Log etc., fit to the calibration curve of multiple spot calibration.According to the absorbance difference of sample (A sample-A is blank), on working curve Try to achieve the content of NY-ESO-1 albumen in sample.

Latex immunoturbidimetry prepared by the present embodiment, for detecting peripheral blood or serum N Y-ESO-1 antigen levels, works as sample Time in product containing NY-ESO-1, the latex microsphere of crosslinking specific antibody can occur coagulation owing to antigen and antibody specific combines, Cause turbidity to increase, change by measuring sample absorbance, reference standard curve, NY-ESO-1 content in quantitative analysis sample, Detection usefulness and enzyme linked immunosorbent assay (ELISA), chemiluminescence immunoassay detection method are close or equivalent.With concentration as abscissa, Absorbance is that ancestor's coordinate draws standard curve as shown in Figure 2, calculates the content of various kinds sample wells.The results are shown in Table shown in 1.

4.2 enzyme linked immunosorbent assays (ELISA) detection NY-ESO-1 antigen

4.2.1 enzyme labelling specific antibody

4.2.1.1 enzyme labelling specific antibody

Use periodate oxidation method labelling.Weigh 5mg horseradish peroxidase (HRP) to be dissolved in 1.0ml distilled water, add Enter the 0.1M NaIO that 0.2ml newly joins₄Aqueous solution, lucifuge stirring 20min under room temperature.Above-mentioned solution is loaded in bag filter, add 50mg Sephadex G-25 dry powder, room temperature mixes, and 3000rpm is centrifuged 5min, takes supernatant.Add 20 μ l 0.2M pH9.5 carbon Phthalate buffer, makes the pH of the HRP of above hydroformylation be increased to 9.0～9.5, adds 10mg IgG antibody [anti-NY-immediately after ESO-1 recombiant protein monoclonal or polyclone IgG antibody (comprise F (ab) '₂Specific Ab fragments) it is dissolved in 1ml 0.01M In carbonate buffer solution], room temperature lucifuge is gently mixed 2 hours.Add the 4mg/ml NaBH that 0.1ml newly joins₄Solution, mixing, then put 4 DEG C 2 hours.Adding the 0.15M pH7.4 PBS solution of 4 times of volumes, being slowly added dropwise while stirring, isopyknic pre-cooling is saturated (NH4)₂SO₄Solution carries out precipitating 1 time, and 10000rpm is centrifuged 30min, removes supernatant, precipitation 0.2ml, 0.15M, pH7.4 PBS Redissolve, load in bag filter, 4 DEG C with 0.15M, pH7.4, PBS, after changing liquid 4-6 time, mistake Sephadex G-200 gel Post, collects the IgG-HRP conjugate peak of brown, and IgG measures (mg/ml)=(OD_280nm-OD_403nm×0.3)×0.62。

The optimal diluted concentration of enzyme labelled antibody determines.With the carbonate buffer solution dilution NY-ESO-1 recombiant protein of 50mM to 1 μ After g/mL, it is coated reaction 4 DEG C of overnight or 37 DEG C of 120min of micropore with 100 μ L/ holes, delays with the phosphate containing 0.05%Tween-20 Rush liquid (PBS) or Tris-HCl buffer (PBS) washs 3 times, close 30min by 10% calf serum room temperature, then be separately added into 100 μ l through volume ratio 1:1000,1:2000,1；4000,1:8000,1:16000,1:32000 with containing 5% calf serum and The anti-NY-ESO-1IgG-HRP that the phosphate buffer (PBS) of 0.05%Tween-20 or Tris-HCl buffer (PBS) dilute Room temperature reaction 60min, washs 3 with the phosphate buffer (PBS) containing 0.05%Tween-20 or Tris-HCl buffer (PBS) Secondary, add the colour developing of tetramethyl biphenyl ammonia (TMB) substrate and observe.It is optimal with the highest dilution hole that obvious blue reacting hole occurs Diluted concentration, typically should be greater than 1:2000.

4.2.1.2 biotin labeling specific antibody

(anti-NY-ESO-1 recombiant protein monoclonal or polyclone IgG antibody (comprise F (ab) ' to 10mg IgG antibody₂Special Property antibody fragment) be dissolved in 1ml 0.01M carbonate buffer solution), add NHS activated biotin, room temperature lucifuge is gently mixed 2 After hour, load in bag filter, 4 DEG C with 0.15M pH7.4 PBS, after changing liquid 4-6 time, 10000rpm is centrifuged 30min and goes Precipitation.IgG measures (mg/ml)=(OD_280nm-OD_403nm×0.3)×0.62。

The optimal diluted concentration of biotinylated antibody determines.NY-ESO-1 weight is diluted with the carbonate buffer solution (CBS) of 50mM Histone, to after 1 μ g/mL, is coated reaction 4 DEG C of overnight or 37 DEG C of 120min of micropore with 100 μ L/ holes, with containing 0.05%Tween-20 Phosphate buffer (PBS) or Tris-HCl buffer (PBS) wash 3 times, with 10% calf serum room temperature close 30min, Be separately added into again 100 μ L through volume ratio 1:1000,1:2000,1；4000,1:8000,1:16000,1:32000 are with containing 5% calf The biotinylation that the phosphate buffer (PBS) of serum and 0.05%Tween-20 or Tris-HCl buffer (PBS) dilute resists NY-ESO-1IgG room temperature reaction 60min, buffers with the phosphate buffer (PBS) containing 0.05%Tween-20 or Tris-HCl Liquid (PBS) washs 3 times, adds the Streptavidin room temperature reaction 15-30min of HRP labelling, with containing 0.05%Tween-20's Phosphate buffer (PBS) or Tris-HCl buffer (PBS) wash 3 times, are eventually adding tetramethyl biphenyl ammonia (TMB) substrate and show Color is observed.To occur that the highest dilution hole of obvious blue reacting hole is optimal diluted concentration, typically should be greater than 1:2000.

4.2.2 be coated and close

Dilute NY-ESO-1 recombiant protein monoclonal with the carbonate buffer solution of 50mM or polyclone IgG antibody (comprises F (ab)’₂Specific Ab fragments) to after 1 μ g/mL, it is coated reaction 4 DEG C of overnight or 37 DEG C of 120min of micropore with 100 μ L/ holes, uses Phosphate buffer (PBS) or Tris-HCl buffer (PBS) containing 0.05%Tween-20 wash 3 times, use 10% calf blood Clear room temperature closes 30min, dries, and can directly carry out next step operation, it is possible to after abundant dehydrate, and sealing is put 4 DEG C and can be grown Phase preserves.

When using biotin-avidin system, after the carbonate buffer solution dilution Streptavidin of 50mM to 1 μ g/mL, It is coated reaction 4 DEG C of overnight or 37 DEG C of 120min of micropore, with the phosphate buffer containing 0.05%Tween-20 with 100 μ L/ holes Or Tris-HCl buffer (PBS) washs 3 times (PBS), close 30min by 10% calf serum room temperature, dry, through being fully dried After, sealing is put 4 DEG C and can be preserved for a long time.

4.2.3 sample-adding

Adding 100 μ L serum samples to be detected, as time quantitative, the concentration the most simultaneously setting up above-mentioned steps 1.4 to prepare is 0, 5,10,25,50,100,250ng/mL NY-ESO-1 recombiant protein gauge orifices, room temperature or 37 DEG C reaction 60min or 4 DEG C overnight.

When using Streptavidin to be coated reaction micropore, each hole is while adding 100 μ L serum samples to be detected, as fixed During amount, the concentration the most simultaneously setting up above-mentioned steps 1.4 to prepare is 0,5,10,25,50,100,250ng/mL NY-ESO-1 restructuring Protein standard hole, adds biotin labeled NY-ESO-1 recombiant protein monoclonal or many prepared by above-mentioned steps 4.2.1.2 Clonal antibody 100 μ L/ hole, mixing.Room temperature or 37 DEG C reaction 60min or 4 DEG C overnight.

4.2.4 enzyme labelled antibody is added

Above-mentioned reaction the micropore phosphate buffer (PBS) containing 0.05%Tween-20 or Tris-HCl buffer (PBS) washing 3 times, finally add the anti-NY-ESO-1 recombiant protein polyclone determined through the optimal diluted concentration of enzyme labelled antibody (comprise F (ab) '₂Specific Ab fragments) or monoclonal antibody IgG-HRP room temperature reaction 60min.

During as used biotin-avidin system and being coated reaction micropore by specific antibody, then add biotinylated Anti-NY-ESO-1 recombiant protein polyclone or monoclonal antibody, room temperature reaction 60min, reaction micropore PBS wash 3 times, then add Enter the Streptavidin room temperature reaction 15-30min of HRP labelling.

4.2.5 colour developing

Above-mentioned reaction the micropore phosphate buffer (PBS) containing 0.05%Tween-20 or Tris-HCl buffer (PBS) washing 3 times, add 100 μ l/ holes tetramethyl biphenyl ammonia (TMB) or o-dihydroxy ammon (OPD) substrate lucifuge colour developing 5-15min Observe.As time quantitative, then add the hydrochloric acid of the 1.8M in 50 μ l/ holes or sulphuric acid to terminate reaction.Each hole is surveyed at 450nm by microplate reader Or the absorbance OD value at 495nm.

4.2.6 result judges

Qualitative: all obvious blue or yellow reaction person occur for the positive, is otherwise negative.

Quantitative: according to the absorbance of anti-NY-ESO-1 gauge orifice, draw standard curve, calculate the content of various kinds sample wells.

Standard curve as shown in Figure 2, calculates the content of various kinds sample wells.The results are shown in Table shown in 1.

4.3 Chemiluminescence immunoassay detection NY-ESO-1 antigens

4.3.1 acridinium ester label specific antibody

Use direct labelling method.10mg IgG antibody (anti-NY-ESO-1 recombiant protein monoclonal or polyclone IgG antibody (comprise F (ab) '₂Specific Ab fragments) it is dissolved in the carbonate buffer solution of 10ml 100mM, pH 9.0 and 160 μ l acridines Ester solution (1mg 2', 6'-dimethyl-carbonyl phenyl 10-methyl-9-acridine formic acid esters 4'-N-succinimide ester trifluoromethyl Sulfonate (being called for short acridinium ester DMAE-NHS) (CAS:115853-74-2) is dissolved in 1.0ml dimethylformamide, acridinium ester DMAE-NHS is purchased from lark prestige Science and Technology Ltd.), IgG antibody and acridinium ester DMAE-NHS mol ratio are 1:4.It is gently mixed mixed Even, room temperature lucifuge is gently mixed 3 hours.Cross Sephadex G-50 gel column (purchased from Pharmacia), with pH 9.0,100mM Phosphate buffer carries out eluting, collects IgG peak, it is thus achieved that traget antibody.IgG measures (mg/ml)=(OD_280nm-OD_403nm×0.3) ×0.62。

The optimal diluted concentration of acridinium ester labeling antibody determines.NY-ESO-1 is diluted with the carbonate buffer solution of pH 9.0,50mM Recombiant protein, to after 1 μ g/mL, is coated reaction micropore with 100 μ L/ holes, places 120min for 4 DEG C overnight or 37 DEG C, delay with phosphate Rush liquid (PBS, pH 9.0) to wash 3 times, close 30min by 10% calf serum room temperature, then be separately added into 100 μ l through little containing 5% The pH 7.4 of Ox blood serum, 50mM phosphate buffer (PBS) with 1:1000,1:2000,1:4000,1:8000,1:16000,1: The traget antibody of 32000 mass ratio dilutions, room temperature reaction 60min, washs 3 times with PBS, adds containing volumetric concentration 0.01%H₂O₂ Tris buffer (pH8.0) 100 μ l, survey each hole luminous intensity with Chemiluminescence Apparatus.With the higher reacting hole of luminous intensity High dilution hole is optimal diluted concentration, typically should be greater than 1:4000.

4.3.2 be coated and close

NY-ESO-1 recombiant protein monoclonal or polyclone IgG antibody is diluted with the carbonate buffer solution of pH 9.0,50mM (comprise F (ab) '₂Specific Ab fragments) to after 1 μ g/mL, it is coated reaction micropore with 100 μ L/ holes, puts for 4 DEG C overnight or 37 DEG C Put 120min, with the pH 9.0 phosphate buffer (PBS) containing volumetric concentration 0.05%Tween-20 or Tris-HCl buffer (PBS) washing 3 times, with 10% calf serum room temperature closing 30min, dry, and through the most dried, sealing is put 4 DEG C and can be protected for a long time Deposit.

When using the detection of biotin-avidin system, dilute Streptavidin with the carbonate buffer solution of pH 9.0,50mM To 1 μ g/mL, it is coated reaction micropore with 100 holes, μ L/ hole, places 120min, with containing volumetric concentration for 4 DEG C overnight or 37 DEG C PH 9.0 phosphate buffer (PBS) or the Tris-HCl buffer (PBS) of 0.05%Tween-20 wash 3 times, little with 10% Ox blood serum room temperature closing 30min, dries, and through the most dried, sealing is put 4 DEG C and can be preserved for a long time.

4.3.3 sample-adding

Add 100 μ L sample to be detected such as serum, blood plasma or other body fluid, as time quantitative, set up above-mentioned steps the most simultaneously The concentration of 1.4 preparations is respectively 0,5,10,25,50,100,250ng/mL NY-ESO-1 recombiant protein gauge orifices, room temperature or 37 DEG C reaction 60min or 4 DEG C overnight.

4.3.4 add acridinium ester label antibody

Above-mentioned reaction micropore with the pH 9.0 phosphate buffer (PBS) containing volumetric concentration 0.05%Tween-20 or Tris-HCl buffer (PBS) washs 3 times, finally adds the acridinium ester label that 100 μ L/ holes determine through above-mentioned steps 4.3.1 The traget antibody of the optimal diluted concentration of antibody, room temperature reaction 60min.

During as used biotin-avidin system and being coated reaction micropore by specific antibody, then add biotinylated Anti-NY-ESO-1 recombiant protein polyclone (comprises F (ab) '₂Specific Ab fragments) or monoclonal antibody, room temperature reaction 60min, the reaction micropore pH 9.0 phosphate buffer (PBS) containing volumetric concentration 0.05%Tween-20 or Tris-HCl delay Rush liquid (PBS) to wash 3 times, add the Streptavidin room temperature reaction 15-30min of acridinium ester label.Above-mentioned reaction micropore is used PH 9.0 phosphate buffer (PBS) or Tris-HCl buffer (PBS) containing volumetric concentration 0.05%Tween-20 wash 3 Secondary.

4.3.5 luminescence is strengthened

Above-mentioned each hole adds 50 μ L substrate solution A and (includes the Tris buffer of 100mM, pH8.0, mass concentration 0.1- The carbamide peroxide (preferably 0.1%, carbamide peroxide is Sigma Co., USA's product) of 0.25% and volumetric concentration 0.01% Triton X-100) and 50 μ L substrate solution B (including the Triton X-100 of 10mM NaOH and volumetric concentration 2%), mixing, Each hole luminous intensity is surveyed at once with Roche E601 chemical illumination immunity analysis instrument (Roche company of the U.S.).

4.3.6 result judges

Quantitative: according to the luminous intensity of anti-NY-ESO-1 recombiant protein gauge orifice, to correct product concentration as abscissa, luminous Intensity is vertical coordinate, draws standard curve, and standard curve as shown in Figure 2, calculates the content of various kinds sample wells.The results are shown in Table shown in 1. The method detection sensitivity 0.01-10ng/mL.

Table 1. testing result of NY-ESO-1 content in 3 kinds of distinct methods clinical serum samples

(unit: ng/mL)

Claims

1. the NY-ESO-1 detection method set up based on specific monoclonal or polyclonal antibody, it is characterised in that described side Method is: prepare anti-NY-ESO-1 antibody with NY-ESO-1 recombiant protein for antigen, and anti-NY-ESO-1 antibody is even with latex microsphere Antibody coupling matter prepared by connection；Then testing sample is mixed with reagent a, 25-37 DEG C of reaction 3-5min, add antibody coupling Thing, 25-37 DEG C of reaction 5-10min, light absorption value at detection 546nm, obtain NY-ESO-1 weight in testing sample according to standard curve Histone concentration；Described anti-NY-ESO-1 antibody is that anti-NY-ESO-1 monoclonal IgG antibody or anti-NY-ESO-1 polyclone IgG resist Body；Described reagent a quality group becomes: 35g/L PEG-4000, bovine serum albumin 5g/L, and solvent is the buffering of pH6.8 Liquid；Described standard curve is prepared as follows: with NY-ESO-1 recombiant protein be antigen prepare anti-NY-ESO-1 specificity resist Body, prepares anti-NY-ESO-1 specific antibody and latex microsphere coupling specific antibody conjugate, then is recombinated by NY-ESO-1 Albumen is diluted to diluent by the 0-250ng/ml gradient concentration buffer containing volumetric concentration 0.1-1% bovine serum albumin, With the buffer containing described bovine serum albumin of nonantigenic composition as blank, then reagent b is diluted with variable concentrations Liquid mixes, 25-37 DEG C of reaction 5-10min, finally adds specific antibody conjugate, 25-37 DEG C of reaction 5-10min, difference Measuring light absorption value at 546nm, with light absorption value as vertical coordinate, in diluent, NY-ESO-1 recombiant protein concentration is as abscissa system Make standard curve；Described anti-NY-ESO-1 specific antibody is anti-NY-ESO-1 specific monoclonal IgG antibody or anti-NY-ESO- 1 specific polyclonal IgG antibody；Described reagent b forms with reagent a.

2. the NY-ESO-1 detection method set up based on specific monoclonal or polyclonal antibody as claimed in claim 1, it is special Levy and be that described anti-NY-ESO-1 monoclonal IgG antibody is the one in IgG1, IgG2a, IgG2b, IgG3 or IgG4；Described anti- NY-ESO-1 polyclone IgG antibody (ab) Han F '₂Specific Ab fragments.

3. the NY-ESO-1 detection method set up based on specific monoclonal or polyclonal antibody as claimed in claim 1, it is special Levy and be that described standard curve is prepared as follows: be that antigen immune rabbit prepares the anti-NY-of rabbit with NY-ESO-1 recombiant protein ESO-1 specific antibody, prepares specific antibody conjugate by anti-for rabbit NY-ESO-1 specific antibody and latex microsphere coupling, will NY-ESO-1 recombiant protein with the buffer containing volumetric concentration 0.25% bovine serum albumin be diluted to 0ng/mL, 5ng/mL, The diluent of 10ng/mL, 25ng/mL, 50ng/mL, 100ng/mL, 250ng/mL, with the buffering containing described bovine serum albumin Liquid is blank, by the diluent of described variable concentrations and reagent b with mix, 25-37 DEG C of reaction 3-5min, be subsequently adding spy Heterogenetic antibody conjugate, 25-37 DEG C of reaction 5-10min, light absorption value at detection 546nm, with light absorption value as vertical coordinate, with diluent Middle NY-ESO-1 recombiant protein concentration makes standard curve as abscissa.

4. the NY-ESO-1 detection method set up based on specific monoclonal or polyclonal antibody as claimed in claim 1, it is special Levy and be that described latex microsphere is with a diameter of 80-400nm, surface active group for-COOH or-NH₂Mass concentration 10% Microsphere latex solution form adds.

5. the NY-ESO-1 detection method set up based on specific monoclonal or polyclonal antibody as claimed in claim 1, it is special Levy and be that described antibody coupling matter preparation method is: (1) latex microsphere activates: by latex microsphere and pH6.5,0.05mol/L MES buffer, water, tween 20 mix, and vortex oscillation mixes；50mg/mL NHS aqueous solution and 10mg/ is added in mixed liquor ML carbodiimide aqueous solution, vortex oscillation 15-45 minute, more ultrasonic 10 times, the most ultrasonic 5 seconds, it is spaced 30 seconds, ultrasonic energy 9000kJ；After ultrasonic end, Sephadex G-25 sephadex column crossed by mixed liquor, washes with water to final volume as initially adding glue 2 times of breast microsphere volume, it is thus achieved that the latex solution of activation；Described latex microsphere with diameter 80-400nm, surface active group for- COOH or-NH₂Mass concentration 10% microsphere latex solution form add；Described latex microsphere with MES buffer volume ratio is 0.4:1, described water and MES buffer volume ratio are 1.5:1, and described tween 20 and MES buffer volume ratio are 1-2:100；Institute Stating NHS aqueous solution with MES buffer volume ratio is 1:10-20, and described carbodiimide aqueous solution and MES buffer volume ratio are 1: 10-20；(2) coupling: anti-NY-ESO-1 antibody is dissolved in pH7.4,100mM PBS and makes antibody-solutions, by antibody After the latex solution mixing that solution and step (1) activate, hybrid reaction 4 hours；Add pH7.2,1.0mol/L glycine buffer Solution hybrid reaction 60 minutes, is centrifuged reactant liquor 20000rpm 30 minutes, abandons supernatant, retains precipitation, resuspended heavy with cleaning mixture Forming sediment, 20000rpm is centrifuged 30 minutes, repeats resuspended and centrifugal 3-5 time, takes the precipitation being finally centrifuged and is antibody coupling matter；Described Cleaning mixture refers to pH7.4,500mM glycine buffer containing volumetric concentration 0.1% bovine serum albumin；The glue of described activation Emulsion volumetric usage is calculated as 0.1-1.0ml/mg with anti-NY-ESO-1 antibody mass.

6. the NY-ESO-1 detection method set up based on specific monoclonal or polyclonal antibody as claimed in claim 1, it is special Levying and be that described testing sample and reagent a volume ratio are 4:125, described testing sample and antibody coupling matter volume ratio are 4:25.

7. the NY-ESO-1 detection method set up based on specific monoclonal or polyclonal antibody as claimed in claim 1, it is special Levy the Tris-HCl buffer that solvent is pH6.8,50mM being described reagent preparation a.

8. the NY-ESO-1 detection method set up based on specific monoclonal or polyclonal antibody as claimed in claim 1, it is special Levy the Tris-HCl buffer that buffer is pH6.8,50mM being described dilution rabbit anti-NY-ESO-1 specific antibody.

9. the NY-ESO-1 detection method set up based on specific monoclonal or polyclonal antibody, it is characterised in that described side By anti-NY-ESO-1 antibody, directly and traget antibody is prepared in acridinium ester DMAE-NHS coupling to method；By testing sample, traget antibody, the end Thing liquid A and substrate solution B mixing carries out luminescence-producing reaction, detects reactant liquor luminous intensity, bent according to NY-ESO-1 recombiant protein standard Line, it is thus achieved that NY-ESO-1 recombiant protein concentration in testing sample；Described anti-NY-ESO-1 antibody and acridinium ester DMAE-NHS material The ratio of amount be 1:3-5；Described anti-NY-ESO-1 antibody is anti-NY-ESO-1 monoclonal IgG antibody or anti-NY-ESO-1 polyclone IgG antibody；Described substrate solution A consists of: mass concentration 0.1-0.25% carbamide peroxide, volumetric concentration 0.01%Triton X-100, solvent is 100mM, pH8.0Tris buffer, and substrate solution B consists of: 10mM NaOH, the Triton of volumetric concentration 2% X-100, solvent is water；Described NY-ESO-1 recombiant protein standard curve preparation method is: NY-ESO-1 recombiant protein is with containing body The Tris-HCl buffer of the 100mM, pH7.4 of volume concentrations 0.25% bovine serum albumin is diluted to 0, and 5,10,25,50,100, The diluent of 250ng/mL, with the buffer containing described bovine serum albumin of nonantigenic composition as blank, is separately added into Described diluent, adds the traget antibody that anti-NY-ESO-1 antibody is directly prepared with acridinium ester DMAE-NHS coupling, adds the end Thing liquid A and substrate solution B carries out luminescence-producing reaction, detects reactant liquor luminous intensity, with NY-ESO-1 recombiant protein concentration in diluent For abscissa, luminous intensity is vertical coordinate, draws standard curve；Described substrate solution A and substrate solution B composition detects with testing sample Substrate solution A and substrate solution B used.

10. the NY-ESO-1 detection method set up based on specific monoclonal or polyclonal antibody as claimed in claim 9, it is special Levy and be that described carbamide peroxide mass concentration is 0.1%.