Deverlopment Of Monoclonal Antibody And Antigen Capture ELISA For Detection Of H7 Subtype Avian Influenza Virus

In this exeriment, 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 the purified highly pathogenic avian influenza virus(AIV) A/FPV/Rostock/34 (H7Nl)(FPV).Two McAb cell strains were determined by hemagglutinin inhibition (HI): 6H4 and 7F6. The subtypes of HA McAbs belong to IgG2a and IgG2b; Both of McAbs are the k strains. Both of McAbs had good specialty by the detection. High-immunized goat serum was achieved by immunizing for three times with purified and inactive A/FPV/Rostock/34 (H7N1)(FPV)mixed with Freund's adjuvant.On the basis of the monoclonal antibody in the laboratory, double-antibodies sandwich ELISA was developed using polyclonal antisera (goat serum against AIV) as capture antibody and H7 specific monoclonal antibody as detecting antibody for detecting H7 avian influenza virus. The optimum conditions were achieved: capture antisera was diluted for 1:1000, McAb was diluted for 1:40000 and the enzyme-labled antibody was diluted for 1:5000. The detecting antibody and conjugate reacted for one and half an hour in the assay.NDV,EDS76,IBV,ILTV,MDV,APV and H1-H16 AIV were detected by the sandwich ELIS and the result showed that there was no crossing-reaction between H7 subtype avian influenza and other avian viruses including HA AIV except H7 by the detecting of the antigen capture ELISA. The detection method of H7 AIV had excellent specialty.The sensitivity of the sandwich ELISA was two times better than that of HA but not better than that of egg inoculation.Many clinical samples infected by men were detected by the sandwich ELISA under the most optimum conditions. Finally, the heart was defined as aim organ when clinical samples were detected by the ELISA and the cut-off was 0.099.The ELISA kit consisted of coated microplated and all kinds of reactive solution and were kept at room temperature, 4℃ and -20℃. The detection results showed that kits had bad stability at room temperature. At the same time, the period of validity in 4℃ and -20℃ was over three months.