副溶血弧菌TDH单克隆抗体的研制与性质鉴定

Preparation and identification of monoclonal antibody against TDH produced by Vibrio parahaemolyticus

目的:制备副溶血弧菌耐热直接溶血毒素TDH单克隆抗体,并分析其免疫学特性。方法:用纯化的TDH毒素蛋白作为免疫原免疫BALB/c小鼠,取免疫小鼠脾细胞与骨髓瘤细胞融合,用间接ELISA方法进行阳性细胞筛选,有限稀释法进行亚克隆。并对该单克隆抗体进行了分子量大小、效价、亚类、特异性和亲和力分析。结果:筛选出一株能够稳定分泌TDH单克隆抗体的杂交瘤细胞株命名为T9H4,其免疫球蛋白亚型鉴定为IgG1,亲和力常数可达2.19×109L/mol,并表现出较强的特异性。抗体纯化后效价达1∶1 024 000,SDS-PAGE电泳结果显示单抗蛋白重链分子量约为50 kD和轻链23 kD。结论:成功获得一株能稳定分泌单克隆抗体的杂交瘤细胞,为开发免疫学快速检测方法提供重要实验基础。

Objective：To prepare monoclonal antibody against TDH（thermostable direct hemolysin） produced by vibrio parahaemolyticus and to analyze their immunologic characteristic.Methods：BALB/c mice were immunized with TDH and spleen cells were fused with SP2／0 myeloma cells,then the positive cells were screened via the indirect ELISA and cloned in limited dilution methods.The molecular weight,titer,subsets,specificity,affinity of monoclonal antibody were analyzed.Results：One strain cell named T9H4,which could secrete monoclonal antibody against TDH,had been obtained.Immunoglobulin subtype of antibody was IgG1 with K chain.The affinity constant was 2.19×109 L/mol and the specificity was significantly exhibited with the titer to 1∶ 1 024 000.In the end,SDS-PAGE showed that the antibody of heavy chain and light chain were 50 kD and 23 kD,respectively.Conclusion：One strain cell which could stably secrete monoclonal antibody against TDH had been obtained successfully,which provide immunological rapid detection with important material basis.