CN105467134A - Reagent for prostatic cancer diagnosis and prognostic immunohistochemical detection - Google Patents
Reagent for prostatic cancer diagnosis and prognostic immunohistochemical detection Download PDFInfo
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- CN105467134A CN105467134A CN201511000438.0A CN201511000438A CN105467134A CN 105467134 A CN105467134 A CN 105467134A CN 201511000438 A CN201511000438 A CN 201511000438A CN 105467134 A CN105467134 A CN 105467134A
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- ehd2
- antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57434—Specifically defined cancers of prostate
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
Abstract
The invention belongs to the technical field of biological detection reagents, and particularly relates to a reagent for prostatic cancer diagnosis and prognostic immunohistochemical detection. The reagent is characterized in that an antibody specific to an EHD2 protein of a human is used as a primary antibody or a core antibody; a chemical marker containing fluorescein, enzyme, metal ions or isotope is used as a second general antibody; the expression quantity of a target antigen (peptide and protein) in histocyte and subcellular localization of the histocyte are detected under color developing action and the like; the EHD2 protein is a protein encoded by gene EHD2; the GeneBank international sequence number of the gene EHD2 is: NM_014601; the GeneBank international sequence number of the gene EHD2 encoded protein is: NP_055416. The invention also discloses application of an EHD2 antibody in preparation of prostatic cancer immunohistochemical detection reagents. As the EHD2 protein participates in multi-step regulation for receptor and cell membrane transshipment, and plays an important role in retaining substance transportation and signal conduction in various cells, the EHD2 protein is closely related to diseases such as tumor.
Description
Technical field
The invention belongs to biological detection reagent technical field, be specifically related to a kind of for prostate cancer diagnosis and Index for diagnosis SABC detection reagent.
Background technology
Prostate cancer is one of kinds of tumor threatening the male sex, in American-European countries, is only second to lung cancer, ranks the second of the male cancer cause of the death.Epidemiologic data shows, and prostate cancer morbidity has significant region, race, age differences.Multinomial research also points out that the morbidity of prostate cancer and habits and customs, cooking culture, working pressure, Serum Testosterone Levels and family's medical history etc. have correlativity.China is in the Di Fa district of prostate cancer, but in recent years, the morbidity of the prostate cancer of China male sex is in rising trend, improve with the aging population aggravation of China, quality of life and living-pattern preservation relevant.
The fundamental research of China's prostate cancer is still relatively weak, and now conventional prostate cancer diagnosis method mainly contains: rectal touch, blood-serum P SA, ultrasonic and puncture, imaging examination etc.The presentation that these diagnostic methods all rest on prostate cancer occurs checks using the foundation judged as diagnosis and prognostic, affect by human factors such as Pathology Doctors ' experiences on the one hand, lack the deeper molecule foundation relevant to individual patient pathogenic factors on the other hand.And at molecular level, coherent detection is carried out to prostate cancer will to prostate cancerization diagnosis and precisely medical treatment is significant.
The generation of tumour is relevant with many factors, and each Affected individuals has again different pathogenic factors.The gene order of carrying out at present detects the main ANOMALOUS VARIATIONS understanding tumour from gene mutation level.But, existing large scale sequencing result of study shows, the function mutation rate of nearly all tumor-related gene only accounts for the very fraction in tumour, in most of the cases, the function mutation that other factors particularly really plays the protein of biological action is only the essence of tumour, and its influence factor can be wrapped again protein-contg unconventionality expression, extremely to be modified, Abnormal regulation and abnormal cell location.Therefore, be the mutation situation of these functional protein molecules clear and definite to the heightened awareness of malignancy of tumor essence and the prerequisite of effectively control, immunohistochemistry is the most effective detection means.Immunohistochemistry technology's applied immunology antigen-antibody reaction ultimate principle, by the specific recognition of anti-body to body internal protein molecular antigen, and the colour developing etc. of the second antibody of chemical labeling is used for test set and knits expression and the Subcellular Localization thereof of cell internal object antigen.
In recent years, along with the development of immunohistochemistry technology and the successful research and development of each strain specific antibodies, deepening continuously more importantly to tumor cells mechanism understanding, SABC obtains general accreditation at diagnosing tumor and the using value in discriminating.Although the nonimmune group of change Testing index such as prostate specific antigen PSA, hexokinase Hk2 are widely used in the clinical molecular pathological diagnosis of prostate cancer, to the pathological analysis of prostate cancer and clinical application role very limited, accurate not.In order to improve constantly the treatment level of prostate cancer, the pathogenesis of more deep research prostate cancer, it is revealed and bring in clinical practice to need other molecular indexes, a kind of EHD2 specific antibody disclosed by the invention and SABC reagent thereof are that the SABC molecular pathology of prostate cancer detects and provides a new molecular indexes, will contribute to the accurate diagnosis and treatment of prostate cancer.
The research of the application's project's earlier stage finds that the expression of EHD2 albumen produces mutation with being positioned in prostate cancer, the EHD2 specific antibody of independent research and can detecting this mutation with the SABC reagent that this antibody is core.EHD2 (Epsinhomology (EH)-domain-containingprotein2) is the one of EHD albumen, is a class new membrane transhipment modulin, the EH domain containing high conservative.Participate in the multistep regulation and control of the after birth transhipments such as acceptor endocytosis, it is the requisite important step of whole after birth transporting mechanism, molecule and the key effect such as matter transportation and intracellular signaling is played in various kinds of cell, its imbalance will cause cell signal to reply improper and cell functional disorders, and possible and then diseases induced.Therefore carry out SABC to EHD2 to detect and reflect the alienation essence of tumour and the clinical characteristics of individual patients by more objective and accurate.At present, the SABC reagent prepared by EHD2 specific antibody has no report, and the application of EHD2 antibody in preparation prostate cancer SABC detection reagent also beyond example.
For solving the problem, the invention discloses a kind of for prostate cancer diagnosis and Index for diagnosis SABC detection reagent.
Summary of the invention
For solving the problem mentioned in background technology, the invention discloses a kind of for prostate cancer diagnosis and Index for diagnosis SABC detection reagent.
The technical matters that the present invention solves is achieved through the following technical solutions:
For solve in background technology mention being mainly also based on histopathology level to the differentiation of tumour clinically carries out routine morphological observation to tumor tissues and tumour cell at present, affect larger by human factors such as Pathology Doctors ' experiences, lack the problem of the deeper molecule foundation relevant to individual patient pathogenic factors, the present invention discloses a kind of for prostate cancer diagnosis and Index for diagnosis SABC detection reagent.
The technical matters that the present invention solves is achieved through the following technical solutions:
A kind of for prostate cancer diagnosis and Index for diagnosis SABC detection reagent, be using a kind of antibody special to people EHD2 albumen as primary antibodie or core antibody; Comprise fluorescein, enzyme, metallic ion or isotope as general second antibody using chemical labeling, waited being used for test set and knitting expression and the Subcellular Localization thereof of cell internal object antigen (peptide and protein) by colour developing.
Described EHD2 albumen refers to the albumen of being encoded by gene EHD2, and the international sequence numbering of GeneBank of described gene EHD2 is: NM_014601, and the international sequence numbering of GeneBank of described gene EHD2 encoding proteins is: NP_055416.
Described a kind of antibody special to people EHD2 albumen, it is characterized in that, described antibody capable specific recognition people EHD2 albumen, the amino acid sequence of described recognition site is: 503-SEQIDNO:1-543, the peptide species be made up of this amino acid sequence, its amino acid sequence is SEQIDNO:1.
With the amino acid sequence of described polypeptide for core sequence is modified it, described in be modified to and hold connection halfcystine at described polypeptide N; Described polypeptide after modification is purified carries out immunity to prepare EHD2 antibody and to carry out antigen purification as antigen to animal afterwards.
Described SABC detects reagent, for detecting the expression of EHD2 in prostate cancer tissue and location, it is characterized in that adopting the aforementioned antibody special to people EHD2 albumen as primary antibodie or core antibody.
Enzyme as described general second antibody can be horseradish peroxidase HRP, bone type alkaline phosphatase, swashs
Enzyme or proteinase.
Beneficial effect
Beneficial effect of the present invention is:
The invention discloses the application of a kind of EHD2 specific antibody in preparation prostate cancer SABC detection reagent, the multistep of transporting due to EHD2 albumen participation acceptor and after birth regulates and controls, and in various cell, maintenance matter transportation and intracellular signaling etc. are played a crucial role, closely related with the generation of the disease such as prostate tumor.Therefore carrying out SABC to EHD2 to detect more objective and accurate from the alienation essence of molecular level reflection tumour and the clinical characters of individual patients, meets accurate medical principle and the trend of tumour.
EHD2 antibody disclosed by the invention detects the application in reagent in preparation prostate cancer SABC, using to the special antibody of people EHD2 albumen as primary antibodie or core antibody, preparation prostate cancer SABC detects reagent, the reagent specificity prepared is good, highly sensitive, colour rendering is good, and effectively can differentiate that EHD2 gene in histocyte is at the expression of protein level and location feature, the accurate medical treatment of the individuation for prostate cancer provides new molecule foundation.
Accompanying drawing explanation
Fig. 1 is immune-blotting method result figure.
Fig. 2 is SABC figure under hyperplastic prostate tissue 200 times of mirrors.
Fig. 3 is SABC figure under prostate cancer tissue 200 times of mirrors.
Fig. 4 is SABC figure under prostate cancer tissue 400 times of mirrors.
Embodiment
Below in conjunction with drawings and Examples, the present invention is elaborated
Embodiment: a kind of for prostate cancer diagnosis and Index for diagnosis SABC detection reagent is using a kind of antibody special to people EHD2 albumen as primary antibodie or core antibody; ; Using HRP as second antibody chemical labeling, waited by dilution colour developing and be used for test set and knit expression and the Subcellular Localization thereof of cell internal object antigen (peptide and protein).
Described EHD2 albumen refers to the albumen of being encoded by gene EHD2, and the international sequence numbering of GeneBank of described gene EHD2 is: NM_014601, and the international sequence numbering of GeneBank of described gene EHD2 encoding proteins is: NP_055416.
Described a kind of antibody special to people EHD2 albumen, it is characterized in that, described antibody capable specific recognition people EHD2 albumen, the amino acid sequence of described recognition site is: 503-SEQIDNO:1-543, the peptide species be made up of this amino acid sequence, its amino acid sequence is SEQIDNO:1.
With the amino acid sequence of described polypeptide for core sequence is modified it, described in be modified to and hold connection halfcystine at described polypeptide N; Described polypeptide after modification is purified carries out immunity to prepare EHD2 antibody and to carry out antigen purification as antigen to animal afterwards.
Described SABC detects reagent, for detecting the expression of EHD2 in prostate cancer tissue and location, it is characterized in that adopting the aforementioned antibody special to people EHD2 albumen as primary antibodie or core antibody.
Enzyme as described general second antibody can be horseradish peroxidase HRP, bone type alkaline phosphatase, swashs
Enzyme or proteinase.
1, to the preparation of EHD2 specific antibody
1) experiment material source
New England's White Rabbit is purchased from Shanghai and shines by force biology, polypeptide
CDEEFALASHLIEAKLEGHGLPANLPRRLVPPSKRRHKGSAE customized by Tianjin Purcell Biological Technology Co and with KLH coupling, CNBr activation gel beads, FuShi Freund's complete adjuvant and FuShi Freund's incomplete adjuvant are purchased from Invitrigen company.
2) animal immune
Get 4 monthly age new zealand white rabbit 3,100ug antigen polypeptide is dissolved in 0.2ml0.1MPBS (pH7.2), fully mix with equal-volume FuShi Freund's complete adjuvant, subcutaneous abdomen multi-point injection.First time the immune rear 15th and 29 days, fully mix rear booster immunization with 100ug polypeptide/0.2mlPBS with equal-volume FuShi Freund's incomplete adjuvant respectively.
3) the sero-fast preparation of EHD2
After immunity terminates, one week arteria carotis gets blood, and 37 DEG C leave standstill centrifuging and taking serum after 3 hours.
4) antigen gel beads preparation
The gel beads of CNBr activation soaks 30 minutes in 1mMHCl, with coupling buffer (containing 0.1MNaHCO3pH8.3, and 0.5MNaCl) cleaning, add the ratio hybrid reaction system of 1ml gel in 1mg polypeptide, 4 degree of couplings are spent the night, 1M monoethanolamine soaked after 3 hours uses cleaning fluid 1 (containing 50mMTris, 1MNaCl, pH8.0) and cleaning fluid 2 (containing 50mM glycocoll, 1MNaCl, pH3.5) intersection cleaning eight times, PBS cleans 1 time.
5) purifying of EHD2 antibody
Serum and above-mentioned antigen gel beads mixing by volume for 20:1, add and mix the rear isopyknic PBS of system, mixing centrifugal after 1 hour, gel beads is cleaned with PBS, be associated in the antibody in gel beads with pH3 sodium citrate solution wash-out, adjust pH to 6.5-7.5, obtain the EHD2 antibody after purifying.
2, Western blot is adopted to carry out specific detection to EHD2 antibody
1) experiment material source:
293T cell is purchased from U.S. AmericanTypeCultureCollection (ATCC) company, RPMI1940 nutrient solution, BSA, HRP mark anti-rabbit two are anti-, Lipo2000 transfection reagent, RIPA lysate, BCA protein concentration detect reagent, ECL chemiluminescence detection reagent etc. and be purchased from Invitrogen company, and EHD1, EHD2, EHD3, EHD4 expression plasmid is self-control.
2) cell chulture
293T cell chulture in RPMI1940 nutrient solution, 37 DEG C, 5%CO2 cultivates adherent growth; First discard nutrient solution during passage, then use phosphate buffer (phosphatebufferedsaline, PBS) to wash twice, add 0.05% Trypsin Induced 2 minutes, add nutrient culture media and stop digestion.Cell maintains a good state, and within two days, passes a generation.Add the plasmid and transfection reagent of expressing EHD1, EHD2, EHD3, EHD4 during transfection respectively, collect cell after 2 days and do immunoblot experiment.
3) western blotting method
Various cell is reserved sufficient amount in centrifuge tube, centrifugal rear RIPA lysate cell lysis, boils sample, more centrifugal.After obtained sample, BCA detection reagent is used to carry out the mensuration of protein concentration.Each sample is got 80ug Tot Prot respectively and is carried out SDS-PAGE electrophoresis.After electrophoresis terminates, forward on pvdf membrane by glue setting egg(s) white appliances, room temperature 5% milk closes 1 hour.After washing, with primary antibodie (antibody prepared in embodiment 1 adds PBS and 5%BSA with 1:2000) incubated at room 1 hour.The anti-incubated at room of the anti-rabbit two of then diluting with 1:5000 1 hour.Finally detect with chemiluminescence detection reagent.
4) result: above-mentioned antibody has the specificity of height to EHD2 albumen, shows as and only just has signal for EHD2, and to even other EHD albumen of very high homology can not identify.Testing result is shown in Fig. 1.In figure: sample Ve is empty expression vector, no signal; Sample 1 process LAN EHD1 albumen, no signal; Sample 2 process LAN EHD2 albumen, be a master tape in corresponding molecular weight 70kd position, signal is clear, not other obvious background band; Sample 3 process LAN EHD3 albumen, no signal; Sample 4 process LAN EHD4 albumen, no signal.To sum up, the antibody that the present embodiment provides has strong signal at 70kd place, normal position, and in the equal no signal in other positions, result shows that antibody provided by the invention has good specificity.
3, EHD2 Immunohistochemical detection
1) experiment material source:
Tianjin Tumour Hospital's tumor tissues sample storehouse is taken from prostate cancer section, conventional dewaxing.General two anti-, diaminobenzidine (DAB) substrate, substrate dilution etc. that primary antibodie dilution, horseradish peroxidase (HRP) mark are purchased from Zhong Shan Golden Bridge.
2) SABC detects EHD2 in reagent assembly condition and sample tissue and expresses the detection method with location:
Method key step is: histotomy dewaxing is to water, and antigen retrieval, Endogenous peroxidase blocks, and drips embodiment (1) antibody for preparing as primary antibodie, 4 DEG C of overnight incubation using 1:200.3 each 5min washed by damping fluid.Drip general HRP ELIAS secondary antibody, at room temperature hatch 30 minutes.3 each 5min washed by damping fluid.DAB develops the color, and redyes, basis of microscopic observation staining conditions after dehydration mounting.
3) result:
In non-cancer hyperplastic prostate tissue EHD2 in epithelial nucleus by force painted, slurry or film weak painted (see Fig. 2).In cancerous tissue, EHD2 expression and location occur abnormal, or are almost feminine gender (see Fig. 3); Or nuclear expression weakens and film caryoplasm expression enhancing (see Fig. 4).
Above experimental result shows, adopt and the invention provides the method for immunohistochemical detection that antibody is core, the expression of EHD2 in prostate cancer tissue cell can be detected well and express position, be convenient to location in cancerous tissue cell of from SABC figure direct interpretation EHD2 and expression, and differentiate that the individuation of prostate cancer is heterogeneous on this basis.
Above-described embodiment is only for illustrating technical conceive of the present invention and feature, and not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment done, equivalent replacement etc., all should be included within protection scope of the present invention.
Claims (4)
1. detect a reagent for prostate cancer diagnosis and Index for diagnosis SABC, it is characterized in that, using to the special antibody of a kind of people EHD2 albumen as primary antibodie or core antibody; Comprise fluorescein, enzyme, metallic ion or isotope as general second antibody using chemical labeling, detect histocyte internal object antigen polypeptide and protein expression amount and Subcellular Localization thereof by dilution, coloration; Described EHD2 albumen refers to the albumen of being encoded by gene EHD2; The international sequence numbering of GeneBank of described gene EHD2 is: NM_014601; The international sequence numbering of GeneBank of described gene EHD2 encoding proteins is: NP_055416; Described a kind of antibody special to people EHD2 albumen refers to antibody capable specific recognition people EHD2 albumen, the amino acid sequence of described recognition site is: 503-SEQIDNO:1-543, the peptide species be made up of this amino acid sequence, its amino acid sequence is SEQIDNO:1.
2. as claimed in claim 1 a kind of for prostate cancer diagnosis and Index for diagnosis SABC detection reagent, it is characterized in that, with the amino acid sequence of described polypeptide for core sequence is modified it, described in be modified to and hold connection halfcystine at described polypeptide N; Described polypeptide after modification is purified carries out immunity to prepare EHD2 antibody and to carry out antigen purification as antigen to animal afterwards.
3. as claimed in claim 1 a kind of for prostate cancer diagnosis and Index for diagnosis SABC detection reagent, it is characterized in that, described SABC detects reagent, for detecting the expression of EHD2 in prostate cancer tissue and location, be adopt the aforementioned antibody special to people EHD2 albumen as primary antibodie or core antibody.
4. as claimed in claim 1 a kind of for prostate cancer diagnosis and Index for diagnosis SABC detection reagent, it is characterized in that, the enzyme as described general second antibody can be horseradish peroxidase HRP, bone type alkaline phosphatase, kinases or proteinase.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020115069A1 (en) * | 1997-02-21 | 2002-08-22 | Mia Horowitz | Eh domain containing genes and proteins |
| CN103923212A (en) * | 2014-03-31 | 2014-07-16 | 天津市应世博科技发展有限公司 | EHD2 antibody and application of EHD2 antibody to preparation of immunohistochemical detection reagent for breast cancer |
| CN104945496A (en) * | 2014-03-31 | 2015-09-30 | 天津市应世博科技发展有限公司 | Polypeptide and application thereof in preparing and purifying EHD2-specific antibody |
| CN104945506A (en) * | 2014-03-31 | 2015-09-30 | 天津市应世博科技发展有限公司 | Immunohistochemical reagent for mammary cancer diagnosis and prognosis judgment |
-
2015
- 2015-12-25 CN CN201511000438.0A patent/CN105467134A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020115069A1 (en) * | 1997-02-21 | 2002-08-22 | Mia Horowitz | Eh domain containing genes and proteins |
| CN103923212A (en) * | 2014-03-31 | 2014-07-16 | 天津市应世博科技发展有限公司 | EHD2 antibody and application of EHD2 antibody to preparation of immunohistochemical detection reagent for breast cancer |
| CN104945496A (en) * | 2014-03-31 | 2015-09-30 | 天津市应世博科技发展有限公司 | Polypeptide and application thereof in preparing and purifying EHD2-specific antibody |
| CN104945506A (en) * | 2014-03-31 | 2015-09-30 | 天津市应世博科技发展有限公司 | Immunohistochemical reagent for mammary cancer diagnosis and prognosis judgment |
Non-Patent Citations (2)
| Title |
|---|
| DAVALIEVA,K等: "Proteomics analysis of malignant and benign prostate tissue by 2D DIGE/MS reveals new insights into proteins involved in prostate cancer", 《PROSTATE》 * |
| 李盼等: "EHD2负向调控前列腺癌细胞的增殖与转化能力", 《中国肿瘤临床》 * |
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