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Hepatitis C is an infectious diseases caused by hepatitis C virus (Hepatitis C virus, HCV) infection. Estimated that about 170 million people worldwide infected with HCV, the number of people infected in China is about 37 million. The partial HCV infection will develop into chronic hepatitis, cirrhosis and hepatocellular carcinoma. Thus, in the early diagnosis of HCV is particularly important. The laboratory diagnosis of HCV: Anti-HCV, HCVAg detect recombinant immunoblot (RIBA), HCV RNA detection. Wherein the anti-HCV detection reagent most widely. Currently, the third generation of the mainstream anti-HCV detection reagents using indirect ELISA, indirect ELISA widespread problem of false positives and missed. Compared with the conventional anti-HCV detection method, double antigen sandwich ELISA method can greatly improve the specificity and sensitivity of detection. Not only can detect IgG antibodies, but also be able to detect early IgM antibodies. However, the double antigen sandwich ELISA kit has not yet been successfully developed. The reason is that the HCV antigen is formed, after an enzyme-labeled, the sterically hindered, hindering the binding of the antibody and antigen. Therefore, the direct labeling of antigen used to establish a double antigen sandwich ELISA is not likely to be successful. Thioredoxin (thioredoxins, TRX) is a small protein of 12 kDa molecular weight of the highly expressed in E. coli and highly hydrophilic, and to provide the necessary signals for the soluble expression of the HCV antigens. HCVAg-trx fusion protein, prepared accordingly Ruozai TRX as osteopontin, osteopontin was prepared the monoclonal antibody, labeled with horseradish peroxidase (HRP). Due bridge protein and the presence of monoclonal antibody, the labeled enzyme will no longer hinder the antigen-antibody binding, better able to solve the problem of steric hindrance. The purpose of the topic for the defects existing hepatitis C diagnostic reagents, through the expression, purification TRX (OPN); prepared TRX (OPN), monoclonal antibodies, biological identification. Labeled with horseradish peroxidase (HRP), to lay the foundation for the development of the hepatitis C double antigen sandwich ELISA kit. Method 1. The TRX expression, purification will contain, the TRX gene of E. coli induced by IPTG for 4 hours, cells were collected, sonicated under ice ultrasound, the target protein was purified by affinity chromatography and 12% SDS-PAGE and identification. 2. The hybridoma strain developed to purify TRX as antigen to immunize 8 week-old female BALB / c mice, three times after immunization, fusion of the immune mouse spleen cells with SP2 / 0 myeloma cells in a 6:1 ratio. Selection culture in the HAT selection medium and screened by indirect ELISA positive cells, the limited dilution Cloning and subcloning, Screening of monoclonal cell lines. 3. ,7-10 days after the intraperitoneal inoculation of monoclonal antibodies Hybridoma collected ascites was purified with n-octanoic acid method and ion chromatography, 10% SDS-PAGE identification. 4. Hybridoma and monoclonal antibody identification with colchicine repressor law so that the cells were arrested in metaphase, under a microscope to observe chromosome count, chromosome analysis of the hybridoma cell lines; using indirect ELISA, monoclonal antibody titers monoclonal antibody affinity relative; using indirect ELISA and double immunodiffusion identification of immunoglobulin class and subclass; improvement the prepared enzyme labeled antibody for the determination of the immune activity of the conjugate of sodium iodide; to block inhibition assay for TRX Determination of mAb recognize the epitope. 5. TRX monoclonal antibody and hepatitis C fusion antigen (HCVAg-trx) reaction was measured by indirect ELISA and Western blotting monoclonal antibody specific reaction of fusion with the hepatitis C antigens (HCVAg-trx). Results 1. Expression of the TRX 12% SDS-PAGE analysis of the purification results showed that the target protein has been highly expressed, the better the separation effect, 95% purity. 2. Development of hybridoma cell lines obtained two stable hybridoma cell lines secreting anti-TRX monoclonal antibody, named 2E8, 5D6. Monoclonal antibodies by UV spectrophotometer calculated 2E8, 5D6 mAb concentration were 1.55mg/mL 0.839 mg / mL. 10% SDS-PAGE: The results show that the purified monoclonal antibody samples appeared in the position of a molecular weight of approximately 150 kd size apparent electrophoretic bands, purity 80%, for the purpose of strip. 4. Hybridoma and monoclonal antibody identification (1) 2E8, 5D6 the chromosome average number of respectively 98,102, in line with the the hybridoma chromosome number of approximately equal mouse spleen cells and SP2 / 0 myeloma cells and chromosome number the theoretical value, thus proving that the cells prepared as hybridoma cells. (2) two monoclonal antibody titer ascites are 1:10 to 7; relative affinity analysis showed that: a 5D6 the relative affinity higher than the 2E8. (3) two mAb were IgG class, IgG1 subclass. (4) 2E8 ELISA antibody immune activity reached to 1:100 × 2 ~ 5,5 D6 enzyme-labeled antibody reached 1:100 x 2 ~~ 6. (5) 2E8 and 5d6 two mAb mutual blocking, prove both identification epitope significantly associated. 5. TRX monoclonal antibodies and recombinant hepatitis C antigen (HCVAg-trx) reaction able to with TRX HCVAg-trx specifically combined two monoclonal antibodies, without cross-reactivity with unrelated proteins IFN-γ. Conclusion 1. IPTG induction and purified by affinity chromatography, high purity recombinant TRX. 2. Successful use of hybridoma technology get two stable secreting anti TRX hybridoma cell lines. 3. Success of hybridoma cell lines and purified antibodies were identified. 4. Two monoclonal antibodies are capable of integration with the hepatitis C antigen specificity binding reaction.
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