| Hepatitis C is a kind of disease infected by Hepatitis C virus (HCV). It has been estimated that about 170 million individuals are infected by HCV in the world, and 37 millon in china. Most of the HCV-infected individuals could result in chronic hepatitis, liver cirrhosis, and even hepatocellular carcinoma. Therefore, the diagnosis for HCV is very important.There are some Laboratory diagnosis methods for HCV, such as HCV-antibody assays, HCV antigen assays, RIBA and RT-PCR. but HCV-antibody assays were used most widely. At present, the model of indirect ELISA is used in most third-generation HCV assays, which usually leads to omission and false-positive of test results. Compared with conventional HCV-antibody assays, Double antigen sandwich ELISA system would remarkably raise the sensitivity and specificity of detection. The double antigen sandwich ELISA system, However, has not been established, Since HCVAg could not be bound to anti-HCV if the HCVAg was labelled directly by HRP.Thioredoxins (TRX) is a protein which can be expressed highly in E.coli and has strong hydrophilicity. It provides an essential signal which results in the expression of HCVAg in soluble. According to this, HCVAg-trx was prepared. TRX can be regarded as a bridge-protein. If anti-TRX McAb was prepared and labelled by HRP, HCVAg would be bound to anti-HCV and not be blocked by HRP due to the existence of bridge-protein and anti-bridge-protein McAb.Hence,it can resolve the space-opstical. ObjectiveAiming at disadvantage of the existing HCV-antibody assays, Trx was expressed and purified firstly, then anti-TRX McAb was prepared, identified by biological methods, labelled by HRP. It would lay the foundation for developing double antigen sandwich anti- HCV ELISA kits.Methods1.Expression and purification of TRX: TRX gene was induced by IPTG to express in E.coli. The recombinant protein which contains His tag sequence ,was purified by the metal chelation chromatography, and identified by 12%SDS-PAGE.2. Preparation of hybridoma cell strains: Purified TRX protein was injected intraperitoneally to the 8-week-old female BALB/c mice. After immunizing for three times, spleen cells were collected from immunized mice, and fused with the SP2/0 myeloma cells at the proportion of 6:1. The hybridoma cells were cultured in the HAT selective medium. The positive clones producing McAbs against TRX were screened by indirect ELISA. Positive cells were cloned and subcloned with limiting dilution method.3.Preparation of monoclonal antibody The cell strains were inoculated into the abdomen of mice. After 7-10 days, ascites was collected. Subsequently, ascites was purified by Caprylc acid-Ammonium sulfate protocol and ion exchange chromatography. Purified McAbs were identified by 10% SDS-PAGE.4. Identification of hybridoma cells and monoclonal antibodies Hybridoma cells were depressed by colchicine at metaphase, and observed with microscope. Their Chromosomes were counted ; Antibody titres and relative affinity were detected by indirect ELISA. The type and subtype of immunoglobulin were identified by both indirect ELISA and double immunodiffusion. The McAbs were labelled by HRP using modified method of heptaiodic acid; Immunocompetence of labelled McAbs was analysed. Finally, recognition of anti-TRX McAbs to epitope was examined by repression method.5.Response of anti-TRX McAbs to recombinant HCVAg (HCVAg-trx) : Specifitic response between anti-TRX McAbs and recombinant HCVAg was detected by both Western blotting and indirect ELISA .Results1.Expression and purification of TRX: It was proved by 12% SDS-PAGE that the recombinant TRX protein had been highly expressed and the purity of TRX was 95%.2.Preparation of hybridoma cell strains:Two hybridoma cell strains secreting monoclonal antibodies (McAbs) against TRX were established and named as 2E8 and 5D6 respectively.3.Preparation of monoclonal antibody:Concentration of the 2E8 was 1.55mg/mL and that of the 5D6 was 0.839 mg/mL. The data from SDS-PAGE showed that the purification was up to 80%. Purified McAbs sample was visualized on electrophoregram, which was proved as targed band.4.Identification of hybridoma cells and monoclonal antibodies:(1) Average number of Chromosomes of the 2E8 was 98 and that of the 5D6 was 102 , which were equal to the total Chromosomes number of spleen cells plus SP2/0 myeloma cells.It suggested that the two cell strains were hybridoma cells.(2) Detection of antibody titres indicated that ascites titer of the two McAbs was up to 1×10~7. Analysis of affinity between two McAbs and TRX showed that relative affinity of 5D6 was higher than that of 2E8.(3) Identification of type and subtype of immunoglobulin demonstrated that both of McAbs belong to IgG1.(4) Immunocompetence of the HRP-1abelled 2E8 reached 1:100×2~5and that of the HRP-labelled 5D6 reached 1:100×2~6.(5) Two kinds of McAbs were repressed with each other. It proved that the epitopes recognized by two McAbs were correlative.5.Response of anti-TRX McAbs to recombinant HCVAg(HCV-trx): Two kinds of anti-TRX McAb obstained could react to TRX and HCV-trx without cross-reaction to IFN-γ. Conclusion1.TRX gene could be induced by IPTG to express in E.coli and the purified recombinant TRX protein was obtained.2.Two hybridoma cell strains secreting monoclonal antibodies (McAbs) against TRX were established successfully.3.Hybridoma cell strains and purified McAbs had been identified successfully.4.Specifitic combination between the two McAbs and HCV-trx was confirmed by both Western blotting and indirect ELISA.
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