抗粉尘螨主要变应原Der fⅡ单克隆抗体的制备与鉴定

Preparation and identification of monoclonal antibodies against Der f Ⅱ allergen

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 Dermatophagoides farinae and to characterize their properties,BALB/c mice were immunized with purified recombinant Der fⅡallergen 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 fⅡ 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 fⅡallergen were obtained,which were determined as IgG2a subtype and good ascites titers.The five McAbs against Der fⅡ allergen all recognized recombinant Der fⅡallergen and native extract from Dermatophagoides farinae.Five monoclonal antibodies against Der fⅡallergen were prepared successfully,which will facilitate establishing detection method of Der fⅡallergen.