【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。更多还原

【Abstract】 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.更多还原