Preparation and identification of monoclonal antibody against bovine IFN-γ

4-6 weeks BALB/c mice were immunized three times with purified rHis-BoIFN-γprotein. Four hybridoma clones secreting antibody against bovine interferon-gamma were obtained by fusing mouse myeloma cells and spleen cells of BALB/c mice. With the purified rGST-BoIFN-γ as detecting antigen,mAbs against BoIFN-γ were prepared and positive hybridoma clones were screened by indirect ELISA.The four mAbs all belonged to IgM subclass with κchain and the ELISA titers of 3 mAbs ascetic fluids were up to 1:104. Western blot analysis suggested four mAbs could only react with the rGST-BoIFN-γ protein, but not react with GST protein.Indirect ELISA showed that four mAbs reacted to rBoIFN-γ, but they didnt reacted to GST protein and cytokine proteins of rGoIFN-α、 rGoIFN-γ and rBoIFN-α. They also showed strong reactivity in indirect immunofluorescence test on the BHK21 cell with transient expressed BoIFN-γ, and suggesting that four mAbs were rBoIFN-γ specific monoclonal antibodies.The result of blocking ELISA detection showed McAb 3D10 could react with natural IFN-γ of bovine peripheral induced blood, and confirmed the McAb had practical value.