Development Of Mcabs Against H9 AIV And Establishment Of Antigen Capture Elisa For Detecting H9 Avian Influenza Virus

In this experiment, six hybridoma cell stains were developed by confusion SP2/0 cells and spleen cells of immunized mouse with AIV FT strain (H9N2). Six positive hybridoma cell stains 1D10,4A7,4E4,4F3,4H12,5B10 were determined by ELISA and HI, which ELISA titers of cell supernatants and ascites were 103,104,105,105,106,105 and 106,107,108,108,109,108 respectively. The HI titers of cell supernatants and ascites of 1D10 and 4E4 were 24,29 and 25,210.The McAbs were specific to H9 AIV and not reacted with H5,H7 AIV. The subtypes of 1D10,4A7,4E4,4F3,4H12,5B10 were IgG2b, IgGl, IgG2a, IgA, IgGl, IgA respectively. Additivity ELISA indicated that 4F3 and 5B10 were corresponding to the same antigen epitope, but the other 4 mAbs were different epitopes. These strains had no neutralization titers against AIV FT strain.4E4 was used as coating McAb for antigen capture ELISA after purified by HiTrapTM Protein G. The optimal working condition was listed as below. (1) capture McAb was diluted to 5μg/well, H9 AIV antigen was diluted to 1:10 and the HRP-labeled polyclonal antibody was diluted to 1:300 (9μg/ml). (2) The optimal condition of coating was 2.Oh in 37℃and then overnight in 4℃.The best blocking reagent was 3% BSA and the optimal blocking time was 1.0h.The reaction time for capturing antigen and HRP-labled polyclonal antibody were both 1.0h. The reaction time for TMB was 15min.The result proved that the H9 AIV antigen capture ELISA had excellent specificity.The sandwich ELISA based on McAbs was experimented with specificity,sensitivity and repetition.The data demonstrated that:(1)NDV,IBDV,IBV,ILTV,FPV and H5,H7 AIV were detected by the sandwich ELISA and the result proved that there was no crossing-reaction between the antigen capture ELISA and other avian viruses and H5,H7 AIV except H9.It proved that the H9 AIV antigen capture ELISA had excellent specificity.(2) The result proved that the H9 AIV antigen capture ELISA had excellent specificity. (3)CV of intra-plate and inter-plate was smaller than 10%.The simple ELISA kits which consisted of coated microplate and reactive solutions were kept at room temperature,4℃and—20℃seperately.The detection results showed that kits were not stable at room temperature and the period of validity was only one month.At the same time,the period of validity at 4℃was 3 months and over 4 months at—20℃.