抗牛IFN-γ单克隆抗体的制备与鉴定

Preparation and identification of monoclonal antibody against bovine IFN-γ

试验以纯化的rHis-BoIFN-γ蛋白为免疫原免疫4～6周龄BALB/c鼠,3次免疫后,取脾细胞与SP2/0骨髓瘤细胞进行融合,用纯化的rGST-BoIFN-γ作为检测抗原,以间接ELISA和有限稀释法进行筛选,并初步鉴定其生物学特性。结果表明,试验共获得4株能稳定分泌抗rBoIFN-γ单克隆抗体的杂交瘤细胞株。抗体亚类均为IgM,轻链为κ链;其中腹水效价2株均达到5×104以上;通过Western blot结果表明,4株单抗均可特异性的结合rGST-BoIFN-γ蛋白,而不与GST标签蛋白反应。间接ELISA试验证明,4株单抗只与rBoIFN-γ反应,而不与GST蛋白和rGoIFN-α、rGoIFN-γ、rBoIFN-α等细胞因子发生交叉反应。通过间接免疫荧光显示,4株单抗均与真核细胞BHK21表达的rBoIFN-γ反应产生强荧光反应,证明了单抗能够与接近天然的BoIFN-γ反应,证实了单抗的特异性。阻断ELISA检测结果表明,3D10株单抗能够与牛外周血诱导产生的天然干扰素发生反应,证实了该单抗具有实际的应用价值。

4-6 weeks BALB/c mice were immunized three times with purified rHis-BolFN-y protein. 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-BolFN-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 K chain and the ELISA titers of 3 mAbs ascetic fluids were up to 1：10^4. Western blot analysis suggested four mAbs could only react with the rGST-BolFN-γ protein, but not react with GST protein.Indirect ELISA showed that four mAbs reacted to rBolFN-γ, but they didnt reacted to GST protein and cytokine proteins of rGolFN-α, rGolFN-γ and rBolFN-α. They also showed strong reactivity in indirect immunofluorescence test on the BHK21 cell with transient expressed BolFN-γ, and suggesting that four mAbs were rBolFN-7 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 MCAS had practical value.