Development And Application Of Monoclonal Antibodies Against The Hemagglutinin Of H9 And H5 Subtype Of Avian Influenza Virus

1 Development of monoclonal antibodies against the hemagglutinin of H9 and H5 subtype of avian influenza virusTo prepare the monclonal antibody (mAb) against the hemagglutinin of H9 or H5 subtype of avian influenza virus (AIV), 8-week-old female BALB/c mice were immunized with the inactivated vaccine of H9 and H5 subtype of AIV. Splenocytes from the immunized mice were fused with Sp2/0-Ag-14 myeloma cells, and positive hybridoma clones were screened by indirect ELISA and hemagglutination inhibition (HI) test. The specificity of the mAbs were characterized by ELISA , HI , indirect immunofluorescence (IF) assay and Western-blot assay. Three hybridoma cell lines named 2H1, 2A3, 1C8 against H9 subtype of AIV were obtained and all these mAbs had the HI activity. The HI liters of these mAbs were 1 X28-1 X213, and the iELISA titers were 1 X 107, 1 X 105 and 5 X 106 respectively. The immunoglobulin subclass of the 3 mAbs were IgG1. Western-blot confirmed that 2H1 mAb recognized the 75KD HA. This mAb reacted with 62 out of 66 H9 subtype of AIV Chinese isolates.Other three hybridoma cell lines named 2G2, 5C3 , 6F6 against H5 sutype of AIV were obtained and 2G2 and 6F6 mAbs had the HI activity. The HI titers were 1X 212, 1 X210, and the iELISA titers were 6X106, 8X105 and 6 X 106 respectively. The immunoglobulin subclass of these mAbs were IgG2a IgG1, IgG1 respectively.These mAbs recognizing H9 or H5 subtype of AIV may provide a useful tool for antigenic analysis, serological diagnosis, the epidemiological survey and the evaluation of vaccine products of AIV.2 Preliminary establishment of indirect DAS-ELISA to detect H9 subtype of avian influenza virusA double-antibody sandwich ELISA(DAS-ELISA) was developed for the detection of the H9 subtype of AIV antigen. In the indirect DAS-ELISA, the coating concentration of chicken polyclonal antibodies against AIV was 1:6 400, the working concentration of primary antibody of mAb 2H1 was 1:8 000. It was shown that the DAS-ELISA was sensitive and specific for the detection of H9 subtype of AIV.