Development and Characterization of Monoclonal Antibodies Against Hemagglutinin of H9 Subtype Avian Influenza Virus

Objective:To develop and characterize monoclonal antibodies(McAbs)against hemagglutinin of H9 subtype avian influenza virus(AIV).Methods:Six to eight-week-old female mice were immunized with H9 subtype AIV.Splenocytes from the immunized mice were fused with Sp2/0-Ag-14 myeloma cells.Hemagglutinin inhibition (HI)test was used to screen hybridoma cells and limited dilution method was performed to subclone the positive clones.Results:After 3～4 cycles of subcloning,six hybridoma cell strains secreting specific McAb were established and designated as 1B2,1C10,1G2,2B7,2E3 and 5E11,respectively.The HI titers in their supernatants were 24～28,and those in ascites were 210～213.Except the immunoglobulin subclass of 1G2 was IgM,all other McAbs were belonged to IgG1.Western blotting analysis confirmed that the McAbs of 1B2,1C10,2B7 and 2E3 only reacted with Mr 75 000 HA protein of H9 subtype AIV.The results of reactivity showed that all the six obtained McAbs only reacted with H9 subtype of AIV but did not react with other subtypes of AIV,Newcastle disease virus and infectious bronchitis virus in HI assay.Conclusion:The results suggested that McAbs specific to hemagglutinin of H9 subtype AIV were developed.These McAbs would be a useful tool for AIV surveillanc,rapid clinical diagnosis, and antigenic epitope analysis.