抗粉尘螨主要变应原Der fⅡ单克隆抗体的制备与鉴定

摘要

目的 制备并鉴定鼠源抗粉尘螨主要变应原Der fⅡ单克隆抗体(Monoclonal Antibody,McAb).方法 重组Der fⅡ蛋白为抗原免疫BALB/c小鼠,取小鼠免疫脾细胞与NS-1细胞融合.间接ELISA法筛选特异性分泌的杂交瘤细胞.用筛选获得的单克隆细胞株诱生小鼠腹水,蛋白G亲和层析法纯化腹水抗体.利用Ig类与亚类鉴定试剂盒鉴定该单克隆抗体的Ig亚型；通过间接ELISA、Western Blotting方法鉴定该单克隆抗体的特性和交叉性.结果 获得5株IgG2a型鼠抗粉尘螨主要变应原Der fⅡ的单克隆抗体,效价良好.ELISA和Western Blotting分析表明该5株单抗均可识别重组Der fⅡ蛋白和天然粉尘螨提取物.结论 成功制备了5株鼠抗粉尘螨主要变应原Der fⅡ的单克隆抗体,为建立粉尘螨主要变应原Der fⅡ的检测及纯化方法奠定了基础.%In order to prepare monoclonal antibodies from mice against Der f II allergen from Dermatophagoid.es farinae and to characterize their properties, BALB/c mice were immunized with purified recombinant Der f II allergen protein, and the splenocytes of the immunized mice were fused with NS-1 myeloma cells by hybridoma technique. Indirect ELISA was used for screening the hybridoma cell lines which could secrete antibodies against Der f II. The McAbs against Der f fl allergen were purified using affinity chromatography on immobilized Protein G to identified their specificity, subtype, titers and cross-reactvity by ELISA and Western blotting. Five hybridoma cell lines secreting McAbs against Der f fl allergen were obtained, which were determined as IgG2a subtype and good ascites titers. The five McAbs against Der f II allergen all recognized recombinant Der f II allergen and native extract from Dermatophagoides farinae. Five monoclonal antibodies against Der f II allergen were prepared successfully, which will facilitate establishing detection method of Der Ⅱ allergen.