Production and Application of Monoclonal Antibodies against Avian Influenza Virus

H5N1 subtype avian influenza virus was chosen to immunize BALB/c mice in order to produce the monoclonal antibodies(McAbs).The allantoic fluid was inactivated by 0.1% formaldehyde,purified and concentrated by differential centrifugation.The spleen cells of immunized mice were fused with Sp2/0-Ag-14 myeloma cells.Indirect enzyme linked immunosorbent assay(ELISA) and hemagglutinatin inhibition(HI) test were used to screen hybridoma cells.Fourteen McAbs specifically against avian influenza virus(AIV) were obtained.One of these McAbs,A6,had the ability of HI,and could react with the rFPV-H5-HA in the infected chicken embryo fibroblast cell(CEF).Two McAbs,4B3 and F2,were coated and conjugated with horseradish peroxides(HRP).Sandwich ELISA was established with the optimal McAbs to be coated and HRP-conjugated.By using the selected 4B3 for both coating and coniugating of HRP,the allantoic fluids of 175 strains of AIV were tested,the positive rates were above 90%.No cross-reaction was observed in the samples of NDV,IBV,IBDV and EDSV.