CN102401831B - Competitive enzyme-linked immunosorbent assay kit for liver cirrhosis and assay method therefor - Google Patents
Competitive enzyme-linked immunosorbent assay kit for liver cirrhosis and assay method therefor Download PDFInfo
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- CN102401831B CN102401831B CN 201110359482 CN201110359482A CN102401831B CN 102401831 B CN102401831 B CN 102401831B CN 201110359482 CN201110359482 CN 201110359482 CN 201110359482 A CN201110359482 A CN 201110359482A CN 102401831 B CN102401831 B CN 102401831B
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Abstract
The invention discloses the application of a monoclonal antibody hybridoma cell strain AP0105 with the collection number of China general microbiological culture collection center (CGMCC) No.5269 to a competitive enzyme-linked immunosorbent assay kit for liver cirrhosis. The competitive enzyme-linked immunosorbent assay kit provided by the invention is prepared from a monoclonal antibody secreted by the hybridoma cell strain, can specifically detect the liver cirrhosis, can be operated easily, conveniently and rapidly, and is favorable for high-flux clinical detection and low in detection cost.
Description
Technical field
The invention belongs to biological technical field, be specifically related to cirrhosis competitiveness enzyme linked immune assay 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 no longer are 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 using more widely, they have all played certain effect at aspects such as the recurrence of creating operative treatment chance, reduction or minimizing tumour for the patient and transfers, have prolonged 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 are used for responsive, high special serodiagnosis early diagnosis of cancer, high, provide the cancer early warning to generally investigate, and are significant and the 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 to the small liver cancer patient of diameter of tumor 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 significantly improve 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 because specificity or cost benefit aspect are difficult to widespread use in General Survey of Liver Cancer.
Polypeptide resource abundant in the serum is played the part of important role at present major disease diagnosis.Adopt conventional at present clinical means to carry out the polypeptide detection and cause gradually the great attention of academia and the world of medicine, diagnosing tumor technology based on polypeptide is just beginning to the clinical practice high speed development, also among exploitation, the use in conjunction of some polypeptide might provide for the early warning of the major diseases such as tumour innovative breakthrough to the early warning technology of tumour.Polypeptide is different with albumen in the serum in the serum, and its molecular weight is lower than 10000Da, and is simple in structure, changes various.Whether can adopt the clinical means of present routine to detect still in exploration.
At present, polypeptide carries out clinical diagnosis also beyond example in the detection serum.Can serum polypeptide be used for diagnosis or early diagnosis liver cancer can be judged from three aspects.1, serum polypeptide is catabolite or the gene direct coding product of haemocyanin.When especially transforming to tumour when physiological status changes, the metabolism of albumen changes, and causes the 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, there has been the diagnosis of some diseases to adopt polypeptide marker in the practice, 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 used for clinical diagnosis, as, Clinical Chemistry 51:10,1933-1945 (2005) and Clinical Chemitry 53:61,067-1074 (2007) think that the serum polypeptide mark can the undistinguishable tumour of more accurate differentiation protein marker.Nat Methods.2008 Jan; 5 (1): 57-9 thinks ms diagnosis tumour even not worse than MRI.The facilitation that mass-spectrometric technique is brought to life science is universally acknowledged, over past ten years, mass spectrum is being obtained a series of impressive progresses aspect newborn two examinations, the detection in Gene Mutation, and single nucleotide polymorphism (SNP) occurred and analyze 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 the clinical field unique identification thing to exist all the time the deficiencies, the particularly verification and measurement ratio to infantile tumour such as specificity is strong, positive rate is lower not high, indicate that the joint-detection thing must go more, realize detecting simultaneously but suffer from the good means of neither one.The people such as Josep Villanueva were to 32 routine prostate cancers, 21 routine breast cancer and 20 routine carcinoma of urinary bladder serum polypeptide researchs in 2006, 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 that exists in the serum provides abundant mark resource for tumour early warning and early diagnosis, and the use in conjunction of these polypeptide might provide for the early warning of the major diseases such as tumour innovative to break through.Find that 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 early warning, and cirrhosis is the important early warning period of liver cancer.Therefore accurately and fast diagnosing of cirrhosis helped prevention and early treatment liver cancer.
The serum polypeptide group of rising in recent years has been successfully applied to the diagnosis research of several cancers, such 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 preserving number is the application of monoclonal antibody hybridoma cell strain AP0105 in preparation cirrhosis competitiveness enzyme linked immunoassay reagent kit of CGMCC No.5269.
Among the present invention, anti-human primary carcinoma of liver polypeptide marker monoclonal antibody hybridoma cell strain AP0105, 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 that then filters 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 common micro-organisms center (address: No. 3, A, DaTun Road, Chaoyang District, BeiJing City, Institute of Microorganism, Academia Sinica, postcode 100101) preservation, preserving number are CGMCC No.5269.
Wherein, described competitiveness enzyme linked immunoassay reagent kit, it contains enzyme target monoclonal antibody of the present invention, and preferred, the enzyme of described mark can be horseradish peroxidase.
Wherein, described competitiveness enzyme linked immunoassay reagent kit can further contain polypeptide standard items, coating buffer, 5% skimmed milk power, TMB nitrite ion, 2M sulfuric acid and 96 hole elisa plates, and the sequence of described polypeptide is Asn-Leu-Gly-His-Gly-His-Lys-His-Glu-Arg-Asp-Gln-Gly-His-Gly-His-Gln.
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, cheaply enzyme connection detector detection.
The present invention can detect polypeptide antibody and ELSIA technical tie-up simultaneously a plurality of biological indication marks groups, 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.
Description of drawings
Fig. 1 is the ROC curve map of detection cirrhosis serum of the present invention and normal serum.
Embodiment
Following examples are used for explanation 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 the 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 the BALB/C mice splenocyte and the SP2/0 murine myeloma cell merges under the PEG effect;
3) screen by the ELISA method, with limiting dilution assay the hybridoma of the secretory antibody positive is carried out 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 and 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.With the strain of the anti-human primary carcinoma of liver polypeptide marker of this hybridoma cell strain called after monoclonal antibody hybridoma cell, and carrying out preservation on September 27th, 2011 at China Committee for Culture Collection of Microorganisms common micro-organisms center, preserving number is CGMCC No.5269.
The assembling of embodiment 2 cirrhosis competitiveness enzyme linked immune assay kits
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 available 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: 53 parts of the cirrhosis serum of clinical definite, 60 parts of normal serums.
Testing process:
1) get 1 μ g/mL polypeptide standard solution, 100 μ L (coating buffer dissolving), place 96 orifice plates, 4 ℃ of placements are spent the night;
2) abandon coating buffer, clean 6 times with 0.01M, pH7.4 PBST damping fluid 200 μ L, pat dry;
3) skimmed milk power of adding 200 μ L 5%, 37 ℃ leave standstill sealing 1.5 hours;
4) abandon confining liquid, clean 6 times with 0.01M, pH7.4 PBST damping fluid 200 μ L, pat dry;
5) the HRP labeling polypeptide antibody of testing sample and 0.3ug/mL with volume ratio 1: 1 outside plate 37 ℃ in conjunction with 1 hour; Replace testing sample as negative control with PBS simultaneously;
6) add 100 μ L 5) middle binding soln, 37 ℃ left standstill 1 hour;
7) abandon 6) middle solution, clean 6 times with 0.01M, pH7.4 PBST damping fluid 200 μ L, pat dry;
8) add 100 μ LTMB nitrite ions, 37 ℃ of lucifuges are hatched the 15min colour developing;
9) add 50 μ L 2M sulfuric acid, cessation reaction;
10) join the absorbance value (OD value) that detector is measured wavelength 450nm with enzyme.
The result shows such as the ROC curve of Fig. 1 by statistics, and it is OD value＜=0.987 that two groups of cirrhosis serum and normal serums are diagnosed positive criteria, susceptibility reaches 91.4%, specificity reaches 89.7%, illustrates with kit provided by the invention to carry out enzyme linked immunosorbent detection, and specificity is very strong, good reproducibility.
The above only is preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from 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. preserving number is the application of monoclonal antibody hybridoma cell strain AP0105 in preparation cirrhosis competitiveness enzyme linked immunoassay reagent kit of CGMCC No.5269.
2. application according to claim 1 is characterized in that, described competitiveness enzyme linked immunoassay reagent kit contains the monoclonal antibody that enzyme target preserving number is the monoclonal antibody hybridoma cell strain secretion of CGMCC No.5269.
3. according to claim 2 described application is characterized in that, described enzyme is horseradish peroxidase.
4. according to claim 2 or 3 described application, it is characterized in that, described kit also contains 1 μ g/ml polypeptide standard items, coating buffer, 5% skimmed milk power, TMB nitrite ion, 2M sulfuric acid and the 96 hole elisa plates as envelope antigen, and the sequence of described polypeptide is Asn-Leu-Gly-His-Gly-His-Lys-His-Glu-Arg-Asp-Gln-Gly-His-Gly-His-Gln.
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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 |
| CN102998450B (en) * | 2012-11-22 | 2014-10-15 | 北京正旦国际科技有限责任公司 | Immuno-mass spectrometric detection kit for esophagus cancer |
| CN107632154A (en) * | 2017-08-31 | 2018-01-26 | 王明丽 | A kind of HCMV pp65 antigenemias Competitive assays ELISA method detection kit and its detection method |
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| WO2011026036A1 (en) * | 2009-08-28 | 2011-03-03 | Inova Diagnostics, Inc. | Detecting circulating cartilage oligomeric matrix protein in liver cirrhosis |
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