Establishment Of Competitive ELISA Based McAb For Detecting H5 Subtype Avian Influenza Antibodies

In this experiment, several hybridoma cell strains were developed by fusion of SP2/0 mouse myeloma cells with spleen cells isolated from a BALB/C mouse which was immunized with Pcagg-HA recombinant plasmid,VSV- HA recombinant virus and ND-AI Vaccine.Nine McAb cell strains were determined by indirect ELISA coated with the purified AIV DZJ/1100(H5N1), were named 10E2B1,16C11A12,16C11B1,14B5H9,11C7H8,9E1A1,14B4B12,15A9H11 and 12G1B1. The specificial McAbs of AIV (ELISA titers) were all above 1:5000. The subtypes of HA McAbs belong IgG3,IgG3,IgG3,IgM,IgM,IgM,IgM,IgM and IgG2b, all McAbs are theκstrains.On the basis of the monoclonal antibody in the laboratory, competitive ELISA was developed using the purified inactived AIV DZJ/1100 and the HA recombinant protein as coating antigen and H5 specific monoclonal antibody as detecting antibody for detecting H5 subtype serum.The optimal conditions were achieved: coating antigen was diluted to 1:1250, McAb was diluted to 1:500 and the enzyme-labled antibody was diluted to 1:5000. The antibody,McAb and enzyme-labled antibody were incubated for an hour in the assay.NDV,EDS76,IBV,IBDV,ILTV,MDV,APV and H1～H16 AIV were detected by the competitive ELISA and the result showed that there was no crossing-reaction between H5 subtype avian influenza and other subtype avian influenza viruses positive serum and others avian and swine diseases viruses positive serum detecting with the competitive ELISA method. The detection results proved that H5 AIV competitive ELISA method had excellent specialty.