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目的研制能诱导肿瘤细胞凋亡的抗人DR5单克隆抗体(McAb)。方法以可溶性人死亡受体(death receptor,DR)5胞外段免疫小鼠,采用杂交瘤技术制备抗人DIL5的MeAb;MTT方法筛选分泌有细胞毒活性McAb的杂交瘤细胞;亲和层析方法纯化McAb;夹心ELISA法测定McAb亚型; Western blot和斑点ELISA法检测McAb的抗原表位类型;间接ELISA法检测McAb的特异性;流式细胞仪检测FTTC-annexinⅤ／PI双色标记的Jurkat细胞的凋亡率;DNA琼脂糖凝胶电泳测定凋亡细胞的DNA片段化。结果获得1株杂交瘤细胞,其分泌的McAb命名为mDRA-6,为IgG1;其抗原表位类型为构象表位;其特异性识别hDR5,与hFas、hDR4等无交叉反应。mDRA-6对Jurkat细胞具有细胞毒作用;经McAb mDRA-6处理后,Jurkat细胞膜表面高表达丝氨酸磷脂,并导致Jurkat细胞中的DNA片段化。结论mDRA-6是一个具有诱导细胞凋亡活性的新的抗人DR5功能性抗体,在以TRAIL／DR5系统进行肿瘤治疗和探讨DR5的功能结构域研究方面具有广泛应用前景。
Objective To develop anti-human DR5 monoclonal antibody (McAb) that can induce tumor cell apoptosis. Methods Mice were immunized with soluble extracellular death receptor (DR) 5 and hybridoma technology was used to prepare MeAb against human DIL5. MTT was used to screen the hybridoma cells secreting cytotoxic McAb. Affinity chromatography Methods The McAb was purified by sandwich ELISA. The type of McAb was detected by Western blot and dot blot ELISA. The specificity of McAb was detected by indirect ELISA. The flow cytometry was used to detect Jurkat cells stained with FTTC-annexinⅤ / PI The apoptotic rate was determined by DNA agarose gel electrophoresis. Results One hybridoma cell line was obtained. The secreted McAb was named mDRA-6, which was IgG1. The epitope type of the McAb was conformational epitope. It specifically recognized hDR5 without any cross reaction with hFas, hDR4 and so on. mDRA-6 has cytotoxic effect on Jurkat cells. After McAb mDRA-6 treatment, the surface of Jurkat cells highly expressed serine phospholipid and resulted in DNA fragmentation in Jurkat cells. Conclusion mDRA-6 is a new anti-human DR5 functional antibody with apoptosis-inducing activity. It has broad application prospect in the field of tumor therapy with TRAIL / DR5 system and the study of the functional domain of DR5.