Preparation Of A Monoclonal Antibody Against Chicken Interferon Gamma And Establishment Of Its Quantitative ELISA

Interferon-gamma(IFN-γ), plays an important roles in anti-virus,anti-tumors and immune modulation. The endogenous level of IFN-γis an important index of body's cellular immune status. The quantitative determination of IFN-γconcentrations has very significant theorical and practical values in immune mechanism research,immune function assay,the efficacity evaluation of vaccine,transplant operation,hypersensitive reaction and intracellular pathogen diagnosis.In this study, BALB/c mice are immunized three times with recombinant chicken interferon-gamma(rChIFN-γ) expreesed in E.coli. According to standard procedure hybridomas are produced by fusing. The positive clone is screened by indirect ELISA and limitingdilution, detecting antigen was recombinant chicken interferon-gamma(rChIFN-γ) expreesed in sf9 cell. Ascites McAb is prepared with hybridoma cell of rChIFN-γand valency is measured. Specificity between antigen and antibody is analyzed by Western-blot and McAb neutralization test;labeling IgG of McAb with HRP,FITC and biotin. Determining valency of three labeling McAb. Based on the conclusion of determining the concentration of rChIFN-γprokaryotic expression and titrating antiviral of sf9 cells, to establish the quantitive ELISA for ChIFN-γ. The IFN-γwas produced by stimulating splenic lymphocyte of SPF chicken and its surface staining are performed using labled McAb with FITC.Through above experiments, hybridoma cell strain which stably secretes McAb of anti- ChIFN-γis obtained and it was named G1 strain, its asctes valency was 1:1×105. The specificity between antigen and antibody was certificated by Western-blot and McAb neultralization test. The separated valencies that labled to IgG with HRP, FITC and biotin reached 1:5000, 1:100, 1:5000. Establishing quantitive ELISA between purified McAb and rChIFN-γof prokaryotic expression, regression equation is Y=2.2742*X+0.105. regression coefficient R is 0.975. Determining minimum detected concentration is 15ng/ml. The coefficient variations of batch inner and interassay coefficient of variation are all less than 10%. The method of quantitive ELISA was established using rChIFN-γexpressed in sf9 cells. Its regression equation is Y=0.1164*X-0.1281, regression coefficient R is 0.9539. The beginning of ICC demonstrates that positive rate of ChIFN-γis 1.71% by stimulus chicken splenic lymphocyte with 10ug/ml ConA. The property of McAb anti-ChIFN-γis stabilize and asctes valency is high . The valencies that labled to IgG with HRP, FITC and biotin are reached effective valencies. The standard curve that rChIFN-γand McAb reflect good linear correlation. Then they have significant reproducibility and stability . that labled McAb with FITC can react with natural ChIFN-γ. The test of ICC demonstrated that labled McAb with FITC can gain specific reaction with natural ChIFN-γ.