| Avian leukosis is one of the serious diseases of poultry industry in china, and AB subgroup avian leukemia caused huge economic losses to local chicken strains. ALV-A and ALV-B is difficult to distinguish, so there is a great significance to screen one monoclonal antibody to distinguish ALV-A and ALV-B for purification and control of A/B subgroup avian leukosis virus.We use the ALV-B positive hybridoma prepared by our laboratory, and then further sub clone and screening to it. There were 4 subs cloning experiments, using the kit of A/B subgroup avian leukemia virus antibody and Screened by the sheep anti-mouse HPR conjugated. Then make an analysis of IFA to the positive hybridoma cell line. Next, take a classification and identification to the Monoclonal antibody which has been screened, by double antibody sandwich method to test subgroup. We also explore the application of the Monoclonal antibody by making neutralization with ALV-B. In the vitro neutralization test,we neutralize the virus and the Monoclonal antibody, then inoculate them into CEF cell and test p27 by ELISA. In the vivo neutralization test, we set a group of virus-antibody neutralization and a group of inoculating virus. The Monoclonal antibodies is neutralized by the virus after dilution, and then infect the chickens used in the experiment. The chickens in the group of inoculating virus were injected the ALV-B viral liquid, three weeks later, we fed several antiviral drugs to them, and then compared effect with the group of virus-antibody neutralization. At last we analyze results of the neutralization of Monoclonal antibody and ALV-B to the indicators of the chickens, including the change of ALV-B antigen, ALV-B antibody, growth performance of chickens, immune organs, pathology of tissue, blood index as well as the appraisal of PCR of ALV-B.The positive hybridoma cells were cultured and we found that they could secrete stably.The antibody titers were 24. Two monoclonal antibodies A9 and G5 were selected. Results showed that the G5 strain could combine with ALV-A and ALV-B with fluorescence by IFA,and the A9 strain could combine with ALV-A, ALV-B, ALV-J with fluorescence IFA. Two monoclonal antibodies A9 and G5 were both IgM type. The result of P27 test was negative,when the monoclonal antibodies combined with ALV-B, demonstrating the monoclonalantibodies have a good neutralizing effect. Chickens were inoculated with ALV-B, which was neutralized with the monoclonal antibodies. Results showed that the antigen-positive were not detected in the first there weeks, but ALV-AB antibody positive rate was as high as 83%. The P27 could be detected on the forth weeks, and the antigen positive rate was as high as 25%,poison attack highest group antigen positive rate of 44%, indicating that antibodies to the virus in the body during and after antibody positive have a higher rate, and challenged group comparison showed antigen-positive rate. Group growth performance of chickens’ weight in antibody neutralization group was significantly higher in the fourth week than the attack group and the control group. The immune organ index of the thymus, spleen and bursa in attack group had a significant difference between the control group and neutralization group.There were no obvious lesions in tissue sections poison attack group. In the role of the blood test, antibody neutralization and control groups similar changes in the indicators, white blood cells, and lymphocytes; the exchange of other antiviral drugs group chicken blood test indicators was significantly lower. PCR results showed that the virus was identified ALV-B strains.In summary: The positive hybridoma cell strains can be combined with ALV-B, but can not distinguish between A/B subgroup. It was found that Chickens in neutralization group still can infect ALV-B but the infection rate was lower than that in Poison attack group by the neutralization test, the results showed that monoclonal antibody and virus neutralization may have certain immune protection. |
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