Establishment And Application Of Hybridoma Cell Lines Secreting Monoclonal Antibodies Against The Hemagglutinin (HA) Of Avian Influenza Virus (AIV)

The low pathogenic AIV of H9N2 and H5N2 subtypes were grown in chicken embryo eggs and purified by differential centrifugation and density centrifugation. Eight-week Balb/c mice were immunized according to Kazuhiro's produce. Two hybridoma named 3E4, 2A4 were generated by fusion of SP20 myelomas and splenocytes from immunized mice and cloned. The two monoclonal antibodies(McAb) produced was screened by ELISA and HI. One of 3E4 against H5N2 subtype HA mouse ascetic fluid(HI 2⁷, ELISA 12800) was purified(protein concentration:7.46mg/mL) and proved to react with only H5N2 AIV in HI and ELISA tests. The other 2A4 against H9N2 subtype mouse ascetic fluid(HI 2¹⁶, ELISA 256000) was purified (protein concentration: 14.9mg/mL) and proved to react with only 4 H9N2 subtype strains in HI and ELISA tests. Both of them didn't react with H1N1, H3N2SIV, NDV, IBDV, EDSV and IBV in cross-reaction of HI and ELISA. The result indicated that the two McAbs were H5N2 and H9N2 subtype-specific.According to the antigenic variation and the pandemic condition of H9N2 subtype AIV in China.It shown that the variant of H9N2 subtype AIV HA was often and universal. Four monoclonal antibodies named 2A4, B4, F6 and H6 react with 25 H9N2 subtype strains from different areas of the whole nation between 1998 and 2005 by the hemagglutination-inhibition (HI)tests. It was sure that the 25 strains purified by filling and limited dilution and identified by HI cross-reaction with anti-sera and RT-PCR were H9N2 subtype. The result from the HI tests with four McAbs indicate that the 25 strains could be divided into two groups. The first group, the 3^rd and the 18^th.strains, have no reaction to the 4 McAbs. Nevertheless, the second group, the rest of 23 strains, reacted to the 4 McAbs strongly. It revealed that the hemagglutination antigenic site of the 3^rd and 18^th H9N2 subtype AIV bounding to the McAbs has varied or lost.,which need further study.