抗人补体膜攻击复合物新抗原单克隆抗体的制备及鉴定

Preparation and identification of monoclonal antibody against neoantigens of human complement membrane attack complex

目的 研制针对人补体膜攻击复合物 (MAC)新抗原的特异性单克隆抗体。方法 在兔红细胞表面组装人MAC ,分离纯化RBC膜蛋白。以初步纯化的MAC免疫Balb c小鼠 ,取免疫小鼠脾细胞与SP2 0骨髓瘤细胞融合 ,采用免疫斑点杂交的方法分别以纯化的单一补体成分C5、C6、C7、C8、C9和MAC同时进行阴性和阳性筛选。结果 获得了 2株仅与MAC反应而不与各单体成分反应的单克隆抗体。进一步以体外组装的补体终末复合物进行鉴定 ,证实所获单克隆抗体可特异性识别MAC复合物中C9分子暴露出的新抗原。经ELISA法测定 ,2株杂交瘤细胞的培养上清液和腹水效价分别为 1× 1 0 -3 ,1× 1 0 -3和 1× 1 0 -6,1× 1 0 -7;单克隆抗体的重链均属小鼠IgG1亚类 ,轻链均为κ型。结论 获得了 2株特异性识别MAC新抗原的单克隆抗体。

Objective To prepare monoclonal antibody (McAb) against neoantigen of the human complement membrane attack complex (MAC). Methods MAC was deposited on rabbit erythrocyte by incubating NHS and rabbit RBC together at 37 ℃ for 30 min. MAC was purified from RBC membrane and used as immunogen. Hybridomas were prepared by fusing myeloma cell line SP2/0 and Balb/c murine spleen cells which had been immunized with primarily purified human MAC. Clones which reacted with MAC but not purified C5-C9 were selected as positive clone. Results Two strain of McAbs, designated as 3A6 and 4C4, were obtained which only reacted with MAC but not purified C5-C9 in spot immunoblot assay. The titers of two McAbs in culture supernatants and ascites fluid were 1×10 -3 , 1×10 -3 和1×10 -6 , 1×10 -7 , respectively. Both McAbs were IgG1 kappa isotypes.Conclusion Two strains of McAb that are against neoantigens on C9 in MAC are obtained.