Preparation Of Monoclonal Antibodies Against The BovIFN-γ And Development Of Sandwich ELISA For BovIFN-γ Detection

IFN-γ was produced mainly by activated Th₁ cells, CD8⁺ cells and NK cells. Besides its antiviral and antitumor activity, IFN-γ has immune regulating functions, such as to activate macrophages, enchance the expression of MHC I and MHC II moleculars, and enhance antigen processing. Therefore, IFN-γ plays a vital role in immune control and immunopathology. It was reported that extragenetic IFN-γ not only can be used as a medicine in treatment of infectious disease and tumor, but also as a vaccine adjuvant to enhance the effect of vaccine. At the mean time, the amount of endogenous IFN-γ could reflect the cell-mediated immune function to some extent. Consequently, antigen specific IFN-γ response could be used as an index of the cell-mediated immune function against specific pathogens. The immuno-detection of IFN-γ, based on specific anti-IFN-γ monoclonal antibody, was a qualitative and/or quantitative method to measure IFN-γ in samples. This method has been applied to fundamental research, clinical immunology, vaccine evaluation, organ transplant, allergy, detection of intracellular pathogen infection and so on. Meanwhile, IFN-γ and its monoclonal antibody also have a broad application in veterinary medicine. Some foreign scientists reported that recombinant bovine IFN-γ (rBovIFN-γ) and its monoclonal antibody have been used for the research of bovine diseases, and in many developed contries, antigen specific bovine IFN-γ assay has been used to detecte several infectious bovine deseases, especially used to detecte bovine tuberculosis. Nevertheless, a few researches were reported in China. This study cloned and expressed recombinant bovine IFN-γ, produced monoclonal and multiclonal antibodies against bovne IFN-γ, and established a sandwich indirect ELISA to detect bovine IFN-γ.1. Expression and identification of BovIFN-γ in E. coliBovIFN-γ mRNA was isolated from whole blood culture mediam after stimulated by PHA. Then BovIFN-γ cDNA was amplificated by RT-PCR using BovIFN-γ specific primers and was inseted to prokaryotic expression vector pET-28a (+). After sequencing, pETBovIFN-γ was transformed to competent E. coli BL21 cells. A 22kDa protein was expressed after IPTG treatment. The concentration of IPTG and the time of IPTG treatment were optimized.2. The production and identification of rBovIFN-γ McAbBy using purified recombinant BovIFN-γ as antigen and lymphocyte hybridoma technique, four hybridoma strains were generated to stably secrete monoclonal antibodies against rBovIFN-γ, named A7, A10, G6, G10, whose immunoglobin subsets were all...