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

Highly pathogenic avian influenza (HPAI) is an extremely contagious disease of poultry leading to high morbidity and mortality caused by some H5 and H7 subtypes of influenza A viruses. At present, the hemagglutination inhibition(HI) test, a convenient method, was widely used in the diagnosis of AIV infection. In this study, the monoclonal antibodies against hemagglutinin of H5-AIV were developed for the HI test.The 8-week-old female BALB/c mice were immunized with subtype H5-AIV SHC and JSC strains, which were isolated from Shanghai in 2000 and Jiangsu in 2002. Spleen cells collected from immunized mice were fused with SP2/0-Ag-14 myeloma cells after the fourth immunization, Hemagglutinin inhibition(HI) test was used to screen hybridoma cells and limited dilution method was performed to subclone the positive clones. After three cycles of subcloning, six McAbs against the hemagglutinin of H5-AIV were obtained, two of which against SHC H5-AIV, and were designated as 1C10 and 5H10; four of which against JSC H5-AIV, were designated as 2D2, 2C8, 3D3 and 3D11, respectively. The obtained McAbs were proved to have high HI and ELISA titers. By using the immunoglobulin subtypes kit, 2D2, 3D 11 and 1C10 were identified to be IgG₁, 2C8 and 5H10 to be IgG₃ and 3D3 to be IgG₂a. The specificity test suggested that the six McAbs were specific to AIV-H5, but not reacted with other subtypes ofAIVs, NDV, IBV, EDSv-76. The neutralization test indicated that all the six McAbs all reacted specifically with HA of H5-AIV.We choosed 50 H5-AIV isolates from different places of China to react with the six McAbs by the HI test. The HI profile of 2C8 and 3D 11 were similar and the reaction rate was as high as 94% for 2C8 and 3D11, whereas they were 90% and 74% for 2D2 and 3D3, respectively. ThelC10 and 5H10 reacted only with the three strains which did not react with the other 4 McAbs. These results showed that the two McAbs, 2C8 and 3D11, could combine with 1C10 or 5H10 to replace the chicken serum in epidemiological investigation of H5 AIV.