Establishment And Biological Properties Of Hybridoma Cell Lines Secreting Anti-AIV(H₉N₂) Idiotypic Antibodies

In the studies, the Anti-AIV (H9)-idiotypic monoclonal antibody was prepared with the hybridoma technology, the Specificity test and Competitive Inhibition test against the Monoclonal antibody (anti-idiotypic antibody) were made, and in vitro Neutralization Test was made in MDCK cell lines.The SPF chicken embryo was incubated with the Standard AIV subtype-H9N2 and got the allantoid fluid. The antigen was purified with Surcose density gradient centrifugation after the step by step centrifugation. According to the floating density scope (1.19-1.25g/cm³)of the virus, it is determined that the density gradient of the surcose is 30-60%; according to the diameter scope of AIV (80-120nm, the average value is 100nm), it is determined that the centrifugation speed is 30,000r/min and the time is 3h. The virus content is 1.638mg/mL with the Spectrophotometry after dialysis.The SPF chickens were incubated with oil-adjuvanted inactivated AIV vaccine, then the high titer serum was collected and the Immunoglobulin G was extracted with the saturated (NH₄)₂SO₄.And the content of the IgG is 4.090mg/mL.With the chicken-derived anti-AIV IgG as the immune, the Freund's complete adjuvant(FCA) vaccine and the Freund's incomplete adjuvant (FIA)vaccine were prepared to incubated female SPF BALB/c mice of 8 weeks. In the first vaccination, the FCA vaccine was inject-ted subcutaneously in multiple locations on back, and the FIA vaccine was used in the second and the third vaccination. The interval of vaccination was 10 days. The IgG was vaccinated before 3 days of cell fusion (injectedintraperitoneally, 0.25mL).The feeder cell was prepared with the mice spleeocytes and celiac macrophages. The spleeocytes of vaccinated BALb/c mice and the myoloma cells NS-1 were fused under the effect of PEG (MW 4,000) with the presence of the feeder cells. They were cultured in the HAT-1640 complete medium in the CO2 (5%) incubator.The indirect ELISA, in which the covering antigen was rabbit-derived Anti-AIV antibody IgG, was applied to detect the hybridoma cell culture supernatant and select fused cell excreted common idiotypic epitope antibody of the chicken and rabbit. Therefore, the hybridoma cell lines excreted the desire Monoclonal antibody were prepared. The pro-test demonstrated the optimum value of the covering antigen is 102ug/mL.Three strains of hybridoma cells that could excrete specific monoclonal antibodies, which were named 2HiDi> 2HiG^ 3H2D7, respectively, were detected by indirect ELISA. The titer of the antibody in the three strains cells culture supernatant is T4, and the titer of the antibody from the ascites was 1:3200. The results demonstrated that the monoclonal antibody could react specifically with the chicken serum against the AIV (H9N2).In the competitive ELISA inhibition test about the anti-idiotypic monoclonal antibody.the coveringantigen was AIV(H9N2),the chicken-derived anti-AIV(H9N2) serum and the different degree dilution of the anti-idiotypic monoclonal antibody were put in the pores, the second antibody was rabbit-derived anti-chicken IgG enzyme labeled antibody and the color-making regent was OPD. And the results showed that the monoclonal antibody could inhibit the combination of AIV with the chicken-derived anti-AIV IgG, and the capacity of the inhibition decreased with the decrease of the monoclonal antibody concentration.The in vitro neutralization test was made in the MDCK, and according to the results based on Reed-Muench method, the median protection dose (PD50 ) of chicken-derived serum against anti-idiotypic monoclonal antibody to MDCKis IO'^/IOuL; the median protection dose (PD50) of chicken-derived serum agianst AIV inactivated vaccine to MDCK is 10"16/10uL. The results demonstrated that the anti-idiotypic monoclonal antibody vaccine and the antibody derived the SPF chicken incubated with ATV inactivated vaccine all could protect MDCK from the virus infection and the protective capacity of the former was stronger than the latter .The neutralization value of the former was 1.6 times than that of the latter.