Preparation and identification of monoclonal antibodies against bovine IFN-γ

To study the applications of monoclonal antibody(McAb) against bovine IFN-γ(BoIFN-γ) in immunology and disease diagnosis,4-6-week-old BALB/c mice were immunized for three times with purified rHis-BoIFN-γ protein,then mouse myeloma cells and the spleen cells of the BALB/c mice were fused.Twelve hybridoma clones secreting antibody against rBoIFN-γ were obtained by screening with the indirect ELISA and 3-5 times subcloning.Twelve McAbs were belonging to IgG isotype,in which five McAbs were belonging to IgG1 isotype,three McAbs were belonging to IgG2a isotype,four McAbs were belonging to IgG2b isotype.The results revealed that the twelve hybridoma cell strains could secret the McAbs against BoIFN-γ stably in vitro.Western-blot and indirect ELISA showed the twelve McAbs reacted to rBoIFN-γ.They also showed strong reactivity in indirect immunofluorescence assay on the BHK-21 cell with transient expressed BoIFN-γ,it suggested that the twelve McAbs were rBoIFN-γ specific McAbs.ELISA showed that,3C2 and 1H4,1G6 and 1G4 were for the different one;1A10,1B2,2B9,3C4,1C1 and 1G6,1G4 and 3C2,1A10 and 1G8 were for the same epitope.