抗人DcR3单克隆抗体的研制及其鉴定

抗人 DcR3 单克隆抗体的研制及其鉴定【摘要】 目的: 制备抗人 DcR3单克隆抗体, 并鉴定其特异性。方法: 纯化的 His DcR3融合蛋白免疫小鼠, 应用淋巴细胞杂交瘤技术制备抗人 DcR3 mAb 并进行纯化, 纯化后的抗体经 Western blot 和ELISA 方法鉴定其特异性、 Ig 亚型和效价。结果: 获得 5 株稳定分泌抗 DcR3 mAb 的杂交瘤细胞, 均属 IgG1 亚型, 其腹水抗体效价为 1×10-5～1×10-7, Western blot 显示 5 株细胞分泌的 mAb 均可识别 DcR3 蛋白, 其中 1 株可与 SW480 细胞成分反应。结论: 成功建立稳定分泌抗人 DcR3 mAb 的杂交瘤细胞株, 其分泌的抗体特异性强、 效价高, 为讨论 DcR3 在组织中的表达、 分布及研制 ELISA 试剂盒奠定基础。【关键词】 DcR3 单克隆抗体 制备 [Abstract] AIM: To prepare monoclonal antibodies(mAbs) against human DcR3 and identify their characterization. METHODS: BALB/c mice were immunized with purified His DcR3 protein, and mAbs against DcR3 which prepared by hybridoma technique were purified and identified by their specificity, subtype, titers via ELISA and Western blot. RESULTS: Five hybridoma cell lines secreting mAbs against human DcR3 were obtained, which were determined as IgG1 subtype and ascites titers of five mAbs against DcR3 reached 1×10-5-1×10-7. Five mAbs were proved to recognize His DcR3 protein specifically, one of which (1B1) could recognize SW480 cell. CONCLUSION: mAbs against DcR3 with high titers and specificity have been prepared and purified successfully, which laid a foundation for the study of DcR3 expression, distribution in tissu and development of ELISA kit. [Keywords]DcR3; monoclonal antibody; preparation 诱骗受体 3(decoy receptor 3, DcR3, 又称 TR6)为近年新发现的一种可溶性肿瘤坏死因子受体(TNFR)超家族成员, 可竞争性结合肿瘤坏死因子(TNF)成员 LIGHT、 FasL 及 TL1A[1, 2], 但并不介导细胞凋亡, 在细胞的生长、 分化、 死亡以及免疫调节等方面发挥重要作用, 与肿瘤、 自身免...