Preparation Of Monoclonal Antibodies Against Porcine Interferon-γ And The Development Of Quantatitive Sandwich-ELISA

Interferon-gamma(IFN-γ), produced mainly by activated T cells, activated natural killer cells and macrophages, plays an important roles in anti-virus, anti-tumors and immune modulation.The endogenous level of IFN-γ is an important index of body's cellular immune status.The quantitative determination of IFN-γ concentrations has very significant theorical and practical values in immune mechanism research, immune function assay, the efficacity evaluation of vaccine, transplant operation, anaphylactoid reaction and intracellular pathogen diaglosis. In this study, BALB/c mice were immunized three times with recombinant porcine interferon-gamma(rpIFN-γ) expreesed in E.coli, Hybridomas were produced by fusing spleen cells from the immunized BALB/c mouse with SP2/0 myeloma cells according to standard procedure.The positive clone was screened by indirect ELISA and limiting dilution. Finally, we obtained a hybridoma which can screte specific monoclonal antibody against porcine IFN-γ stably. The monoclonal antibody was named B11 strain. Meanwhile, laying hens were immunized four times with the same antigen and the eggs were collected. The partial purified egg yolk antibodies (IgY) were obtained by chloroform extraction and ammonium sulphate precipitation from egg yolk. The antigen specific IgY were biotinylated after separated by rpIFN-γ-Sepharose 4B immunoaffinity chromatography. It was measured that the egg yolk antibodies were labeled with an average of 4.37 biotin molecules per molecule of IgY by means of HABA assay. A sandwich ELISA was developed using the monoclonal antibody(B11) purified by rpIFN-γ-Sepharose 4B immunoaffinity chromatography as a capture antibody and the biotinylated IgY as a detecting antibody. The minimunm detectable concentration by this ELISA was 7.8ng/mL. A standard curve was generated by plotting the average absorbance(OD₄₅₀) obtained for each standard concentration on the vertical axis vs. the corresponding recombinant porcine IFN-γ concentration on the horizental axis.