| It is estimated that 170 to 200 million people are chronically infected with HCV worldwide, which up to 3% of the total population, approximately 3 to 4 million new infected cases occurring each year. The majority of HCV-infected individuals may developed into chronic hepatitis, which led to liver cirrhosis and hepatocellular carcinoma(HCC) after a long progress. Following with AIDS, hepatitis C is becoming a pathogen which threat human health seriously. So far, we understand HCV and hepatitis C poorly. Progress in the pathogenic mechanisms of HCV, the development of a vaccine against HCV and therapy of hepatitis C has been hampered by the lack of an appropriate small animal model. Although chimpanzees are easily infected with HCV, these animals are rare expensive and thus inappropriate for use in research. Therefore, the establish of a small animal model, which easy to breed and with low hold cost, would be of great importance in facilitating progress of HCV research. The HCV susceptibility on tree shrew has been proved by previous researches. However, there are still some impediments in the detection of antibody in tree shrew serum, which is important for evaluation on immune response. There are not commercial kits or methods yet. It is necessary to isolate and purify immunoglobulin from this animal, and then prepare polyclonal antibody and monoclonal antibody.Here, tree shrew serum IgG was purified by caprylic acid-ammonium sulfate precipitation, and then purified by Sephacryl-S200 gel filtration and DEAE-Sephadex A-50 anion-exchange chromatogram. The result of SDS-PAGE revealed that the IgG had been further purified by each step of purification. The molecular weight of IgG is 160kD, and that of heavy chain is 53.5kD, while that of light chain is 28.4kD. The purified IgG was used as the antigen to immunize rabbit, then the anti-IgG polyclonal antibody was prepared. The determined titer of anti-serum with double immunodiffusion was 1:8, and the highest titer of the anti-serum determined by indirect ELISA procedure was 1:25600. An indirect ELISA for detecting serum antibody was established. The optimized reaction system is 1.25μg/ml of coated antigen in working concentration, and the optimum diluted concentration of the tested serum is 1:1000, and the optimum diluted concentration of the enzyme-linked antibody is 1:8000. The result of western blotting showed that the antibody can be used to detect antigen-specific IgG and had immunological activity by reaction with heavy chain and light chain of tree shrew serum IgG. An anti-IgG polyclonal antibody with high titer and high specificity was successfully obtained by immunization of rabbits with tree shrew IgG.The 6-week-old female BALB/c mice were immunized subcutaneously and intraperitoneally with 100μg purified IgG, which emulsified in Freund's complete adjuvant for the first primary injection and in Freund's incomplete adjuvant for the three enhanced immunization. Injections were given at 2-week intervals, followed by an intravenous dose of the antigen without adjuvant. On the third day after the final immunization, splenocytes were isolated and fused with a HAT-sensitive mouse myelomane SP2/0, by the polyethylene glycol (PEG) method. Highly purified Polyethylene 1500(PEG-1500) was used for cell fusion. Cell fusion was performed in a 50ml centrifuge tube. Mingle splenocytes and myeloma cells (5:1) into the centrifuge tube, and then instill PEG-1500 scrupulously at a certain dose. Put the mixed cells into the cell culture wells that had been paved with feeder cells, and selected-culture with HAT. The screening of positive cells with indirect ELISA is going on.In summary, the methods of immunoglobulin purification from tree shrew serum, and preparation of the polyclonal antibody on rabbit have been established. Meanwhile, we have prepared purified IgG and high titer polyclonal antibody. Monoclonal antibody was also prepared preliminary. All of these provide a useful tool for tree shrew antibody detection, will be the basis for HCV vaccine evaluation.
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