Establishment and Characterization of Hybridoma Cell Secreting Monoclonal Antibody Against Hemagglutinin of the Pandemic Swine-Origin Influenza Virus

Objective:To prepare hybridoma cell secreting monoclonal antibody(McAb)against hemagglutinin(HA) of swine-origin influenza virus(S-OIV).Methods:BALB/c mice were immunized twice at regular intervals of 2 weeks with 50 and 25μg of recombinant HA antigen of S-OIV derived from Bac-to-Bac system.Thereafter,mice were boosted intravenously with 12.5μg of recombinant antigen 3 days before the fusion of splenocytes with Sp2/0 cells.Hybridoma culture supernatants were screened and evaluated by ELISA.Positive clones were checked for isotype and McAbs were confirmed by Western blotting analysis using several seasonal influenza virus stocks and ELISA coating HA peptides of S-OIV or H5N1.Results:We obtained six strains of stable hybridoma cells secreting McAb against HA of S-OIV.Three strains(1E12,3F12 and 1C11)of which recognized to HA peptides of S-OIV by ELISA,not to that of H5N1.Furthermore,when analyzed by Western blotting,McAb 1B3 only reacted with HA of S-OIV,not with that of seasonal H1 and H3 influenza viruses.Conclusion:Six strains of specific and stable hybridoma cells secreting McAb against HA of S-OIV have been obtained,which provide useful tools for diagnosis and analyzing of pandemic S-OIV H1N1 infection.