CN102520175B - Competitive enzyme-linked immunosorbent assay (ELISA) kit for liver cancer polypeptide marker antigen - Google Patents
Competitive enzyme-linked immunosorbent assay (ELISA) kit for liver cancer polypeptide marker antigen Download PDFInfo
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- CN102520175B CN102520175B CN201110359486.4A CN201110359486A CN102520175B CN 102520175 B CN102520175 B CN 102520175B CN 201110359486 A CN201110359486 A CN 201110359486A CN 102520175 B CN102520175 B CN 102520175B
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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
- G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
- C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
- C07K14/8139—Cysteine protease (E.C. 3.4.22) inhibitors, e.g. cystatin
Abstract
The invention discloses a competitive enzyme-linked immunosorbent assay (ELISA) kit for a liver cancer polypeptide marker antigen. The competitive ELISA kit contains a monoclonal antibody and a competitive polypeptide standard substance, wherein the monoclonal antibody has a preservation number of CGMCC No.5269 and is secreted by an anti-human primary hepatic carcinoma polypeptide marker monoclonal antibody hybridoma cell strain AP0105, the competitive polypeptide standard substance is a polypeptide marker in a sample to be tested, and the polypeptide has the sequence of Asn-Leu-Gly-His-Gly-His-Lys-His-Glu-Arg-Asp-Gln-Gly-His-Gly-His-Gln. The monoclonal antibody provided by the invention has strong specificity specific to the polypeptide marker antigen; and the detection method of the polypeptide marker antigen is simple, convenient and rapid in operation steps and is favorable for clinical detection; and the high-flux and low-cost detection of an enzyme linked analyzer can be carried out.
Description
Technical field
The invention belongs to biological technical field, be specifically related to liver cancer polypeptide marker antigenic competition enzyme-linked immunologic detecting kit and detection method thereof.
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, as the important supplement of 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 the small liver cancer patient who diameter of tumor is less than 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 can be 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 polypeptide detection and cause gradually the great attention of academia and the world of medicine, 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 with albumen difference 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 be used for diagnosis or early diagnosis liver cancer can judge from three aspects.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 likely breaks through for the early warning of the major diseases such as tumour provides innovative.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
The object of the present invention is to provide liver cancer polypeptide marker antigenic competition enzyme-linked immunologic detecting kit and detection method thereof.
Liver cancer polypeptide marker antigenic competition enzyme-linked immunologic detecting kit provided by the invention, its contain deposit number be CGMCC No.5269 anti-human primary carcinoma of liver polypeptide marker monoclonal antibody hybridoma cell strain AP0105 secretion monoclonal antibody and and testing sample in the polypeptide standard items of polypeptide marker competition, the sequence of described polypeptide is Asn-Leu-Gly-His-Gly-His-Lys-His-Glu-Arg-Asp-Gln-Gly-His-Gly-His-Gln.
This wherein, described hybridoma cell strain is that (sequence is as the polypeptide of Asn-Leu-Gly-His-Gly-His-Lys-His-Glu-Arg-Asp-Gln-Gly-His-Gly-His-Gln take polypeptide marker antigen, 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.
Wherein, described monoclonal antibody is monoclonal antibody linked with peroxidase, and preferred, the enzyme of described mark can be horseradish peroxidase.
Liver cancer polypeptide marker antigenic competition enzyme-linked immunologic detecting kit of the present invention also can further contain coating buffer, 5% skimmed milk power, TMB nitrite ion, 2M sulfuric acid and 96 hole elisa plates.
Detection liver cancer polypeptide marker antigen detection method 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 1-1.5 μ g/mL polypeptide standard solution 100 μ L (coating buffer dissolving), be placed in 96 orifice plates, 4 ℃ of placements spend the night or 37 ℃ place 2 hours;
2) abandon coating buffer, with 0.01M, pH7.4 PBST damping fluid 200-300 μ L cleaning 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 cleaning 5-7 time, pat dry;
5) the HRP labeling polypeptide antibody of testing sample and 0.3 μ g/mL with volume ratio 1: 1 outside plate 37 ℃ in conjunction with 0.5-1 hour; Using PBS, replace testing sample as negative control simultaneously;
6) add 80-120 μ L 5) middle binding soln, 37 ℃ of standing 0.5-1 hour;
7) abandon 6) middle solution, with 0.01M, pH7.4 PBST damping fluid 200-300 μ L cleaning 5-7 time, pat dry;
8) add 80-120 μ LTMB nitrite ion, 37 ℃ of lucifuges are hatched 15min colour developing;
9) add 30-70 μ L 2M sulfuric acid, cessation reaction;
10) with enzyme connection detector, measure the absorbance value (OD value) of wavelength 450nm.
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 multiple 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, the hybridoma of the secretory antibody positive is carried out to clone purification with limiting dilution assay;
4) filter out the hybridoma of 10 strains for 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 competitiveness enzyme linked immune assay 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) polypeptide standard solution: the coated solution of 1 μ g/ml synthetic peptide 5;
3) coating buffer: the carbonate buffer solution of 0.1M pH9.6;
4) 5% skimmed milk power: 5g/100ml skimmed milk power;
5) TMB nitrite ion: be century biotechnology company purchased from Beijing health;
6) 2M sulfuric acid;
7) 96 hole elisa plates.
The competitiveness enzyme linked immune detection method of embodiment 3 polypeptide marker antigens
Sample: each 60 parts of the liver cancer serum of clinical definite, normal serum.
Testing process:
1) get 1 μ g/mL polypeptide standard solution 100 μ L (coating buffer dissolving), be placed in 96 orifice plates, 4 ℃ of placements are spent the night;
2) abandon coating buffer, with 0.01M, the 200 μ L cleaning of pH7.4 PBST damping fluid 6 times, pat dry;
3) add the skimmed milk power of 200 μ L 5%, 37 ℃ of standing sealings 1.5 hours;
4) abandon confining liquid, with 0.01M, the 200 μ L cleaning of pH7.4 PBST damping fluid 6 times, pat dry;
5) the HRP labeling polypeptide antibody of testing sample and 0.3 μ g/mL with volume ratio 1: 1 outside plate 37 ℃ in conjunction with 1 hour; Using PBS, replace testing sample as negative control simultaneously;
6) add 100 μ L 5) middle binding soln, 37 ℃ are standing 1 hour;
7) abandon 6) middle solution, with 0.01M, the 200 μ L cleaning of pH7.4 PBST damping fluid 6 times, pat dry;
8) add 100 μ LTMB nitrite ions, 37 ℃ of lucifuges are hatched 15min colour developing;
9) add 50 μ L 2M sulfuric acid, cessation reaction;
10) with enzyme connection detector, measure the absorbance value (OD value) of wavelength 450nm.
Result by statistics, as the ROC curve of Fig. 1 shows, two groups of diagnosis positive criterias of liver cancer serum and normal serum are OD value <=0.967, susceptibility reaches 94.3%, specificity reaches 95.1%, illustrate and carry out enzyme linked immunosorbent detection with kit provided by the invention, 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 (4)
1. a liver cancer polypeptide marker antigenic competition enzyme-linked immunologic detecting kit, it is characterized in that, contain deposit number and be CGMCC No.5269 anti-human primary carcinoma of liver polypeptide marker monoclonal antibody hybridoma cell strain AP0105 secretion monoclonal antibody and and testing sample in the polypeptide standard items of polypeptide marker competition, the sequence of described polypeptide is Asn-Leu-Gly-His-Gly-His-Lys-His-Glu-Arg-Asp-Gln-Gly-His-Gly-His-Gln.
2. detection kit according to claim 1, is characterized in that, described monoclonal antibody is monoclonal antibody linked with peroxidase.
3. detection kit according to claim 2, is characterized in that, described enzyme is horseradish peroxidase.
4. according to the detection kit described in claim 1～3 any one, it also contains 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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| CN201110359486.4A CN102520175B (en) | 2011-11-14 | 2011-11-14 | Competitive enzyme-linked immunosorbent assay (ELISA) kit for liver cancer polypeptide marker antigen |
| PCT/CN2012/000221 WO2013071678A1 (en) | 2011-11-14 | 2012-02-21 | Liver cancer polypeptide marker antigen competitive enzyme-linked immunosorbent assay kit |
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| CN201110359486.4A CN102520175B (en) | 2011-11-14 | 2011-11-14 | Competitive enzyme-linked immunosorbent assay (ELISA) kit for liver cancer polypeptide marker antigen |
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| CN102520175A CN102520175A (en) | 2012-06-27 |
| CN102520175B true CN102520175B (en) | 2014-04-16 |
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| 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 |
| 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 |
Citations (6)
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| CN1795388A (en) * | 2003-05-26 | 2006-06-28 | 金振宇 | Diagnostic kit for liver cirrhosis comprising an antibody specific for human protooncogenic protein |
| CN101672850A (en) * | 2009-01-05 | 2010-03-17 | 中山大学 | Kit for distinguishing and diagnosing hepatocirrhosis and early liver cancer and application of serum clusterin in preparing same |
| CN201477095U (en) * | 2009-09-02 | 2010-05-19 | 同昕生物技术（北京）有限公司 | Sensitive and fast cirrhosis and liver cancer magnetic bead enzyme-free diagnostic reagent kit and magnetic beads |
| EP2216651A1 (en) * | 2007-10-18 | 2010-08-11 | Miyazaki Prefectural Industrial Support Foundation | Biomarker for diagnosis of liver disease |
| WO2011026036A1 (en) * | 2009-08-28 | 2011-03-03 | Inova Diagnostics, Inc. | Detecting circulating cartilage oligomeric matrix protein in liver cirrhosis |
| 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 |
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- 2011-11-14 CN CN201110359486.4A patent/CN102520175B/en active Active
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- 2012-02-21 WO PCT/CN2012/000221 patent/WO2013071678A1/en active Application Filing
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1795388A (en) * | 2003-05-26 | 2006-06-28 | 金振宇 | Diagnostic kit for liver cirrhosis comprising an antibody specific for human protooncogenic protein |
| EP2216651A1 (en) * | 2007-10-18 | 2010-08-11 | Miyazaki Prefectural Industrial Support Foundation | Biomarker for diagnosis of liver disease |
| CN101672850A (en) * | 2009-01-05 | 2010-03-17 | 中山大学 | Kit for distinguishing and diagnosing hepatocirrhosis and early liver cancer and application of serum clusterin in preparing same |
| WO2011026036A1 (en) * | 2009-08-28 | 2011-03-03 | Inova Diagnostics, Inc. | Detecting circulating cartilage oligomeric matrix protein in liver cirrhosis |
| CN201477095U (en) * | 2009-09-02 | 2010-05-19 | 同昕生物技术（北京）有限公司 | Sensitive and fast cirrhosis and liver cancer magnetic bead enzyme-free diagnostic reagent kit and magnetic beads |
| 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 |
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