CN102539767B - ELISA (Enzyme-Linked Immunosorbent Assay) kit for detecting polypeptide marker antigen - Google Patents
ELISA (Enzyme-Linked Immunosorbent Assay) kit for detecting polypeptide marker antigen Download PDFInfo
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- CN102539767B CN102539767B CN201110300080.9A CN201110300080A CN102539767B CN 102539767 B CN102539767 B CN 102539767B CN 201110300080 A CN201110300080 A CN 201110300080A CN 102539767 B CN102539767 B CN 102539767B
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- monoclonal antibody
- polypeptide marker
- polypeptide
- marker antigen
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
- C07K16/303—Liver or Pancreas
-
- 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
- G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
Abstract
The invention discloses an ELISA (Enzyme-Linked Immunosorbent Assay) kit for detecting a polypeptide marker antigen. The kit contains a monoclonal antibody of the polypeptide marker antigen; and the monoclonal antibody is secreted by a hybridoma cell strain CGMCC No.5269 of a polypeptide marker monoclonal antibody capable of resisting primary liver cancer. The invention also provides a method for detecting the polypeptide marker antigen. The monoclonal antibody provided by the invention has the advantage of strong specificity aiming at the polypeptide marker antigen; and the method for detecting the polypeptide marker antigen is simple, convenient and rapid in operation step and beneficial to clinical detection, and can be used for carrying out high-flux and low-cost detection of an enzyme linked detector.
Description
Technical field
The invention belongs to biological technical field, be specifically related to detect the ELISA kit of polypeptide marker antigen.
Background technology
Along with the ripe and development of Surgery For Hepatocellular Carcinoma, the methods for the treatment of of liver cancer and means are day by day abundant, and are no longer confined to single operative treatment.Hepatectomy and liver transfer operation remain the main method of liver cancer patient treatment, but minimally-invasive treatment, through technical methods such as skin hepatic arterial chemoembolizations, important supplement as operative treatment, also obtained application more widely, they,, creating operative treatment chance, reduction for patient or reducing the recurrence of tumour and the aspect such as transfer has all played certain effect, have extended patient's life span.Even so, liver cancer patient morbidity concealment, non-evident sympton, most of patients have lost the chance of surgical radical treatment when medical.Therefore, " early detection, early diagnosis, early treatment " is the important measures that reduce cancer mortality.Screening and foundation, for responsive, high special serodiagnosis early diagnosis of cancer, high, provide cancer early warning to generally investigate, and are significant and great market prospect.
Extensively adopt at present detection Serum Alpha Fetoprotein (AFP) to make a definite diagnosis liver cancer (HCC) both at home and abroad, although this method has improved the detection level of HCC, but still have remarkable quantity patient's alpha-fetoprotein not raise, especially diameter of tumor is less than the small liver cancer patient of 3CM, its susceptibility and specificity are lower.In recent years, the research of the application of mark joint inspection in diagnosing tumor obtains very large attention.As significantly improved the positive rate of diagnosing cancer of liver by joint inspection alpha-fetoprotein (AFP), carcinomebryonic antigen (CEA), serum ferritin (SF), alpha-L-fucosidase (AFU).The experiment of the four mark joint inspection primary hepatic carcinomas such as AFP, CEA, SF, CA also obtains good result.But due to specificity or cost benefit aspect, be difficult to widespread use in General Survey of Liver Cancer.
Polypeptide resource abundant in serum is played the part of important role in major disease diagnosis at present.Adopt conventional at present clinical means to carry out the great attention that polypeptide detection causes academia and the world of medicine gradually, diagnosing tumor technology based on polypeptide is just starting to clinical practice high speed development, the early warning technology of tumour is also among exploitation, and the use in conjunction of some polypeptide is likely for the early warning of the major diseases such as tumour provides innovative breakthrough.In serum, polypeptide is different with albumen in serum, and its molecular weight is lower than 10000Da, simple in structure, changes various.Whether can adopt the clinical means of current routine to detect still in exploration.
At present, detect polypeptide in serum and carry out clinical diagnosis also beyond example.Can serum polypeptide can judge from three aspects for diagnosis or early diagnosis liver cancer.1, serum polypeptide is catabolite or the gene direct coding product of haemocyanin.While especially transforming to tumour when physiological status changes, the metabolism of albumen changes, and causes polypeptide respective change, thereby has set up polypeptide---and the correlationship of-disease, therefore, polypeptide can carry out medical diagnosis on disease as albumen.2, in practice, there is the diagnosis of some diseases to adopt polypeptide marker, since 1985, utilized synthetic polypeptide to detect AIDS virus (P24), rubella virus, human cytomegalovirus and rheumatoid disease etc. and be applied to clinical.3, document shows, polypeptide can be for clinical diagnosis, as, Clinical Chemistry 51:10,1933-1945 (2005) and Clinical Chemitry 53:61,067-1074 (2007) thinks that serum polypeptide mark can distinguish the undistinguishable tumour of protein marker more accurately.Nat Methods.2008 Jan; 5 (1): 57-9 thinks that ms diagnosis tumour is even not bad than MRI.The facilitation that mass-spectrometric technique is brought to life science is universally acknowledged, nearly ten years, mass spectrum is being obtained a series of impressive progresses aspect newborn two examinations, detection in Gene Mutation, and occurred that single nucleotide polymorphism (SNP) analyzes professional mass spectrometer, shown the value of mass spectrum aspect clinical diagnosis.
Early diagnosis of tumor kit based on serum polypeptide antigen is one of current early diagnosis of tumor technology of greatest concern.At present in clinical field, unique identification thing exists the deficiencies such as specificity is strong, positive rate is lower all the time, particularly not high to the verification and measurement ratio of infantile tumour, indicates that joint-detection thing must go more, but suffer from the good means of neither one, realizes simultaneously and detecting.The people such as Josep Villanueva in 2006 are to 32 routine prostate cancers, 21 routine breast cancer and 20 routine carcinoma of urinary bladder serum polypeptide researchs, find 14,10 and 58 can be respectively as the tumor-marker polypeptide of prostate cancer, breast cancer and carcinoma of urinary bladder, predictablity rate is 100%.The abundant polypeptide existing in serum provides abundant mark resource for tumour early warning and early diagnosis, and the use in conjunction of these polypeptide is likely for the early warning of the major diseases such as tumour provides innovative to break through.Find after deliberation, sequence is that the polypeptide of Asn-Leu-Gly-His-Gly-His-Lys-His-Glu-Arg-Asp-Gln-Gly-His-Gly-His-Gln is one of important serum polypeptide mark of cancer.
The serum polypeptide group of rising has in recent years been successfully applied to the diagnosis research of several cancers, as breast cancer, oophoroma, prostate cancer etc., what mainly adopt is surface enhanced laser desorption ionization (SELDI) technology and immunomagnetic bead technique, but it is expensive that this system exists, the shortcoming of unfavorable popularization.
Summary of the invention
One object of the present invention is to provide the hybridoma cell strain of a kind of polypeptide marker antigen and carrier protein couplet thing.
Another object of the present invention is to provide the monoclonal antibody being produced by above-mentioned hybridoma cell strain.
The present invention also provides the ELISA kit that detects polypeptide marker antigen.
The application of monoclonal antibody described in the present invention also provides in polypeptide marker antigen detects.
The present invention also provides a kind of method that detects polypeptide marker antigen.
The monoclonal antibody of polypeptide marker antigen provided by the invention is secreted by anti-human primary carcinoma of liver polypeptide marker monoclonal antibody hybridoma cell strain AP0105, it is to take polypeptide marker antigen (sequence is the polypeptide of Asn-Leu-Gly-His-Gly-His-Lys-His-Glu-Arg-Asp-Gln-Gly-His-Gly-His-Gln, called after polypeptide 5) with the conjugate immunogene of carrier protein keyhole limpet hemocyanin (KLH), immune balb/c mice, the hybridoma cell strain then filtering out.Anti-human primary carcinoma of liver polypeptide marker monoclonal antibody hybridoma cell strain AP0105 provided by the invention, in on September 27th, 2011 at China Committee for Culture Collection of Microorganisms's common micro-organisms center (address: No. 3, A, DaTun Road, Chaoyang District, BeiJing City, Institute of Microorganism, Academia Sinica, postcode 100101) preservation, preserving number is CGMCC No.5269.
The ELISA kit of detection polypeptide marker antigen provided by the invention, it contains enzyme target monoclonal antibody of the present invention, and preferred, the enzyme of described mark can be horseradish peroxidase.
The ELISA kit of detection polypeptide marker antigen of the present invention can further contain one or more in coating buffer, 5% skimmed milk power, TMB nitrite ion, 2M sulfuric acid and 96 hole elisa plates.
The method of detection polypeptide marker antigen provided by the invention, comprises step:
(1) collected specimens;
(2) with described kit, detect;
(3) analyzing and testing result.
Preferably, described method specifically comprises the steps:
1) get 5-30 μ L testing sample, add 70 μ L-95 μ L coating buffers, be placed in 96 orifice plates, 4 ℃ of placements spend the night or 37 ℃ place 2 hours; Add 100 μ L coating buffers as negative control simultaneously;
2) abandon coating buffer, with 0.01M, pH7.4 PBST damping fluid 200-300 μ L, clean 5-7 time, pat dry;
3) add the skimmed milk power of 200-300 μ L 5%, 37 ℃ of standing sealing 1-2 hour;
4) abandon confining liquid, with 0.01M, pH7.4 PBST damping fluid 200-300 μ L, clean 5-7 time, pat dry;
5) add 80-120 μ L HRP labelled antibody, 37 ℃ of standing 0.5-1 hour;
6) abandon antibody-solutions, with 0.01M, pH7.4 PBST damping fluid 200-300 μ L, clean 5-7 time, pat dry;
7) add 80-120 μ LTMB nitrite ion, 37 ℃ of lucifuges are hatched 15min colour developing;
8) add 30-70 μ L 2M sulfuric acid, cessation reaction;
9) with enzyme connection detector, measure the absorbance value (OD value) of wavelength 450nm;
10) to detect the ratio of OD value and negative OD value, be greater than 2.1 positive.
Monoclonal antibody provided by the present invention is for the high specificity of polypeptide marker antigen; The simple operating steps of the detection method of polypeptide marker antigen is quick, is beneficial to clinical detection; Can carry out high flux, enzyme connection detector detection cheaply.
The present invention, by polypeptide antibody and ELSIA technical tie-up, can detect a plurality of biological indication marks groups simultaneously, and can be used for large-scale pattern detection, is checking blood serum designated object and the ideal tools that is applied to clinical detection.
Accompanying drawing explanation
Fig. 1 is the ROC curve map of detection liver cancer of the present invention and normal serum.
Embodiment
Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.
The preparation method of embodiment 1 polypeptide marker antigen monoclonal antibody
1) adopt the synthetic polypeptide 5 of coupling carrier albumen KLH as immunogen immune BALB/C mice; Its full length sequence is: Asn-Leu-Gly-His-Gly-His-Lys-His-Glu-Arg-Asp-Gln-Gly-His-Gly-His-Gln.
2) detect tail drop of blood degree after 2 weeks, reach 1: 1000 above after, get BALB/C mice splenocyte and SP2/0 murine myeloma cell merges under PEG effect;
3) by ELISA method, screen, with limiting dilution assay, the hybridoma of the secretory antibody positive is carried out to clone purification;
4) filter out 10 strains for the hybridoma of synthetic peptide 5, be surprised to find that a wherein strain susceptibility higher (1ng/well), amplifying cells system, preparation purified monoclonal antibody ascites, detect tire (1: 200000), the titre (0.0005 μ g/ml) of monoclonal antibody and hypotype and identify (IgG2b) etc., obtain the monoclonal antibody specific of anti-polypeptide 5.By the strain of the anti-human primary carcinoma of liver polypeptide marker of this hybridoma cell strain called after monoclonal antibody hybridoma cell, and at China Committee for Culture Collection of Microorganisms's common micro-organisms center, carrying out preservation on September 27th, 2011, preserving number is CGMCC No.5269.
The assembling of the ELISA detection kit of embodiment 2 polypeptide marker antigens
1) monoclonal antibody specific of the anti-polypeptide 5 of the embodiment 1 of 1.25 μ g/ml horseradish peroxidase-labeled;
2) coating buffer: the carbonate buffer solution of 0.1M pH9.6;
3) 5% skimmed milk power: 5g/100ml skimmed milk power;
4) TMB nitrite ion: be century biotechnology company purchased from Beijing health;
5) 2M sulfuric acid;
6) 96 hole elisa plates.
The ELISA detection method of embodiment 3 polypeptide marker antigens
Sample: each 160 parts of the liver cancer serum of clinical definite, normal serums.
Testing process:
1) get the clinical serum of 5 μ L, add 95 μ L coating buffers, be placed in 96 orifice plates, 4 ℃ of placements are spent the night; Add 100 μ L coating buffers as negative control simultaneously;
2) abandon coating buffer, with 0.01M, pH7.4 PBST damping fluid 200 μ L, clean 6 times, pat dry;
3) add the skimmed milk power of 300 μ L 5%, 37 ℃ of standing sealings 2 hours;
4) abandon confining liquid, with 0.01M, pH7.4 PBST damping fluid 200 μ L, clean 6 times, pat dry;
5) add 100 μ L HRP labeling polypeptide antibody, 37 ℃ standing 1 hour;
6) abandon antibody-solutions, with 0.01M, pH7.4 PBST damping fluid 200 μ L, clean 6 times, pat dry.
7) add 100 μ LTMB nitrite ions, 37 ℃ of lucifuges are hatched 15min colour developing;
8) add 50 μ L 2M sulfuric acid, cessation reaction;
9) with enzyme connection detector, measure the absorbance value (OD value) of wavelength 450nm;
10) to detect the ratio of OD value and negative OD value, be greater than 2.1 positive.
By statistics, as the ROC curve of Fig. 1 shows, susceptibility reaches 80.8% to result, and specificity reaches 96.2%, illustrates with kit provided by the invention and carries out enzyme linked immunosorbent detection, and specificity is very strong, reproducible.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, do not departing under the prerequisite of the technology of the present invention principle; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (5)
1. an anti-human primary carcinoma of liver polypeptide marker monoclonal antibody hybridoma cell strain AP0105, its deposit number is: CGMCC No.5269.
2. the ELISA detection kit that contains the monoclonal antibody of hybridoma cell strain AP0105 secretion claimed in claim 1.
3. detection kit according to claim 2, is characterized in that, described monoclonal antibody is monoclonal antibody linked with peroxidase.
4. detection kit according to claim 3, is characterized in that, described enzyme is horseradish peroxidase.
5. according to the detection kit described in claim 2～4 any one, it also contains one or more in coating buffer, 5% skimmed milk power, TMB nitrite ion, 2M sulfuric acid and 96 hole elisa plates.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
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| CN201110300080.9A CN102539767B (en) | 2011-09-29 | 2011-09-29 | ELISA (Enzyme-Linked Immunosorbent Assay) kit for detecting polypeptide marker antigen |
| PCT/CN2012/000220 WO2013044575A1 (en) | 2011-09-29 | 2012-02-21 | Elisa kit for detecting polypeptide marker antigen |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201110300080.9A CN102539767B (en) | 2011-09-29 | 2011-09-29 | ELISA (Enzyme-Linked Immunosorbent Assay) kit for detecting polypeptide marker antigen |
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| CN102539767A CN102539767A (en) | 2012-07-04 |
| CN102539767B true CN102539767B (en) | 2014-03-12 |
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| CN102998452B (en) * | 2012-11-22 | 2014-09-10 | 北京正旦国际科技有限责任公司 | Preparation method for polypeptide marker Pep5 enzyme-linked immunosorbent assay (ELISA) kit |
| CN102998451B (en) * | 2012-11-22 | 2015-02-04 | 北京正旦国际科技有限责任公司 | Detection kit related to liver disease process and application of kit |
| CN103319571B (en) * | 2013-05-30 | 2015-04-08 | 北京正旦国际科技有限责任公司 | Nasopharyngeal carcinoma radiotherapy-sensitive polypeptide marker SPG03 and ELISA kit thereof |
| CN112521455B (en) * | 2020-12-08 | 2022-09-02 | 长春理工大学 | Polypeptide for detecting bladder cancer antigen protein specificity and application thereof |
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| WO2003089616A2 (en) * | 2002-04-22 | 2003-10-30 | Fred Hutchinson Cancer Research Center | Soluble mic polypeptides as markers for diagnosis, prognosis and treatment of cancer and autoimmune diseases or conditions |
| WO2009039421A1 (en) * | 2007-09-20 | 2009-03-26 | University Of Louisville Research Foundation, Inc. | Peptide biomarkers predictive of renal function decline and kidney disease |
| CN102181400A (en) * | 2010-10-15 | 2011-09-14 | 北京蛋白质组研究中心 | Hybridoma cell strain and anti-HCCLM-6 (Human Hepatoma Cell Line) cell secretory protein monoclonal antibody |
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Patent Citations (4)
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| EP2216651A1 (en) * | 2007-10-18 | 2010-08-11 | Miyazaki Prefectural Industrial Support Foundation | Biomarker for diagnosis of liver disease |
| CN101412756A (en) * | 2008-11-25 | 2009-04-22 | 塔里木大学 | Method for detecting chlorothalonil pesticide residue and detection kit therefor |
| CN101533010A (en) * | 2009-02-27 | 2009-09-16 | 中国人民解放军第四军医大学 | Method for quantitatively testing MG7-Ag in serum |
| CN102062780A (en) * | 2010-11-23 | 2011-05-18 | 北京正旦国际科技有限责任公司 | Polypeptide immunoassay kit and detection method thereof |
Non-Patent Citations (2)
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| CN102539767A (en) | 2012-07-04 |
| WO2013044575A1 (en) | 2013-04-04 |
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Inventor after: Wei Kaihua Inventor after: He Ruiyun Inventor after: Sun Yunbo Inventor after: Hou Liping Inventor after: Zhou Xiaoming Inventor after: Fu Haiyuan Inventor after: Yuan Jian Inventor after: Yang Baoan Inventor after: Zheng Junjie Inventor after: Zhen Bei Inventor before: Wei Kaihua Inventor before: Zhen Bei Inventor before: Zhang Tuo Inventor before: Wang Dongmao Inventor before: Zhou Xiaoming Inventor before: Fu Haiyuan Inventor before: Yuan Jian Inventor before: Yang Baoan Inventor before: Hou Liping Inventor before: Sun Yunbo Inventor before: Huang Yajuan Inventor before: Zheng Junjie |
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Free format text: CORRECT: INVENTOR; FROM: WEI KAIHUA ZHOU XIAOMING FU HAIYUAN YUAN JIAN YANG BAOAN HOU LIPING SUN YUNBO HUANG YAJUAN ZHENG JUNJIE ZHEN BEI ZHANG TUO WANG DONGMAO TO: WEI KAIHUA SUN YUNBO HOU LIPING ZHOU XIAOMING FU HAIYUAN YUAN JIAN YANG BAOAN ZHENG JUNJIE ZHEN BEI HE RUIYUN |
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