抗人GPA~N单克隆抗体的制备及鉴定

目的　制备抗GPA单克隆抗体以建立“GPA体细胞突变分析法”。方法　用O ,N型人红细胞 ,以及纯化人GPAN 免疫BALB/c小鼠 ,经杂交瘤技术制备 ,用细胞凝集、间接免疫荧光筛选 ,用ELISA、Westernblot、酶处理红细胞的间接免疫荧光标记鉴定。结果　获得抗GPAN 单克隆抗体 6A8杂交瘤细胞系。其单抗属lgG3亚类 ,λ型轻链 ,识别和结合GPAN 氨基端 1～ 8位决定位点 ,对糖链依赖。流式细胞分选结果显示 6A8特异结合二甲亚胺固定的人MN和N型红细胞。结论　对分析由于基因突变和糖基化变异所致N ,MN红细胞GPAN 突变有理论研究和应用价值。

Preparation and Characterization of McAb to Human GPAN

Objective To prepare anti GPA McAb for establishing GPA somatic mutation assay.Methods The McAb to human GPA N was prepared with the spleen cells of BALB/c mice immunized with O, N type human erythrocytes and purified GPA N by hybridoma technique, selected by cell agglutination and indirect immuno fluorescence microscopy, and identified by ELISA, Western blot and indirect immuno fluorescence with enzyme treated human erythrocytes.Results A McAb cell strain 6A8(IgG3,λ) recognizing the epitopes at sites 1 8 of GPA N was obtained. The strain was dependent on glycosyl chain. Flow cytometric selection showed that 6A8 specifically bound to MN and N type human erythrocytes fixed with dimethyleneimine.Conclusion The study has theoretical and practical values in analyzing the GPA N mutation caused by gene mutation and glycosylation variation.