Production And Application Of Monoclonal Antibodies Against Avian Influenza Virus

Avian influenza has raised great concerns for its economic importance to poultry industry and potential threat to the public health. In this representation, a highly pathogenic avian influenza virus, A/Chicken/Shanghai/2000(H5Nl), was chosen to immunize BALB/c mice in order to product 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 and limited dilution method was performed to subclone positive clones.Fourteen of McAbs specifically against avian influenza virus (AIV) were obtained, designated as 2B6, 3A5, 4B3, 5A5, 5C2, F2, A6, 1A2, 1A4, ID3, 4A6, 5A2, 6B5 and 6C4 respectively. Two of these 14 McAbs, 2B6 and 5A5, were of IgM subtype. Four McAbs, 4B3, F2, A6 and 6C4, were of IgG2a and the others were of IgG1 subtype. One of these McAbs, A6, had the ability of HI, and could be detected in two samples of 64 H5 subtype AIV tested. Furthermore, A6 could react with the rFPV-H5-HA in the infected chicken embryo fibroblast cell (CEF). It was verified that 11 McAbs could react with H5 subtype AIV and all the rates of reaction were lower than 56% (36/64) in indirect ELISA. Three McAbs, 5A5, 4B3 and F2, could react with H5 and H9 subtype AIV and the rates reacting with H5 subtype AIV were 48% (31/64), 89% (57/64) and 89% (57/64) respectively. And that of H9 subtype AIV were 49% (30/61), 83% (51/61) and 90% (55/61) respectively. Two McAbs, 4B3 and F2, were coated and conjugated with horseradish peroxides (HRP). The specificity of the coated/HRP-conjugated McAbs was detected respectively by sandwich ELISA and direct ELISA respectively.Sandwich ELISA was established with the optimal McAbs to be coated and HRP-conjugated. By using the selected 4B3 for both coating and conjugating of HRP, the allantoic fluids of 64 strains of H5 subtype AIV, 61 strains of H9 subtype AIV, 36 strains of H3 subtype AIV, 6 strains of H1 subtype AIV, 2 strains of H4 subtype AIV, 2 strains of H6 subtype AIV, 2 strains of H1 1 subtype AIV were tested, the positive testing rates were 98(63/64), 90% (55/61), 97% (35/36), 100% (2/2), 100% (2/2), 100% (2/2) and 100% (2/2) respectively. No cross-reaction was observed in the samples of NDV, IBV, IBDV and EDSV, additionally, the lowest detection concentration of AIV was 0.4g/ml.