抗人胱蛋白酶抑制剂C单克隆抗体的制备及鉴定  

Production and characterization of the monoclonal antibodies against cystatin C

作　　者：徐杨[1] 杜惠芬[1] 连晓雯[1] 张瑞君[1] 柴丹丹[1] 李克生[1] 

机构地区：[1]甘肃省医学科学研究院医学生物技术研究中心,甘肃兰州730050

出　　处：《中国卫生检验杂志》2016年第17期2436-2438,2442,共4页Chinese Journal of Health Laboratory Technology

基　　金：甘肃省科技厅项目(1204FKCA178)

摘　　要：目的制备抗人Cys-C单克隆抗体并对其生物学特性进行鉴定。方法用基因工程表达的Cys-C抗原免疫BALB/c小鼠,用杂交瘤技术制备抗人Cys-C单克隆抗体,间接ELISA法检测单克隆抗体的效价、亲和力,鉴定单克隆抗体的亚类并检测抗原位点互补关系,SDS-PAGE鉴定抗体纯度,秋水仙素阻抑法鉴定染色体数目。结果筛选出2株能稳定分泌抗人Cys-C单克隆抗体的杂交瘤细胞株,分别命名为2H2、3C4,抗体Ig亚类均为IgG1,亲和常数分别为7.429×10-8、1.95×10-7,2株单克隆抗体抗原位点不重叠,染色体鉴定符合杂交瘤细胞的特性。结论成功制备了特异性的抗人Cys-C单克隆抗体,为Cys-C检测试剂盒的研究开发奠定了基础。Objective To prepare the monoclonal antibody（ m Ab） against cystatin C（ Cys-C） and characterize their biological fuctions. Methods BALB / c mice were immunized with Cys-C antigen expressed by genetic engineering. The m Ab against Cys-C was prepared with hybridoma technique. Indirect enzyme-linked immunosorbent assay（ ELISA） method was used to detect the titers and the affinity of m Abs,and the complementary relationship between the antigen sites was detected; monoclonal antibodies subtypes were identified,and the complementary relationship among antigenic sites were detected. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis（ SDS-PAGE） was used to identify the purity of antibody. The chromosome number was identified with colchicine inhibition. Results Two hybridoma cell strains that could secret Cys-C stably were obtained,named as 2H2 and 3C4,and its antibody subtypes were all IgG1. The affinity constant were 7. 429 × 10^-8,1. 95 ×10^-7. Two antibodies belonged to different antigen determinants. Chromosome identification was consistent with the characteristics of the hybrid cells. Conclusion The monoclonal antibody against Cys-C has been successfully obtained,which established foundations for Cys-C detection kit for research and development.

关 键 词：胱蛋白酶抑制剂C 单克隆抗体 制备 鉴定 

分 类 号：Q784[生物学—分子生物学]