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目的制备抗人DR5单克隆抗体(mAb),鉴定其特性,并进行生物学活性分析。方法以纯化的可溶性DR5(sDR5)免疫Balb/c小鼠,杂交瘤技术制备抗人DR5mAb;运用ELISA、SDS-PAGE电泳方法测定抗DR5mAb与sDR5结合的特性;Ig亚类ELISA试剂盒鉴定抗DR5mAb亚类;间接ELISA法检测腹水mAb效价;流式细胞仪检测肿瘤细胞表面DR5的表达水平;流式细胞仪检测抗DR5单克隆抗体(mAb)诱导肿瘤细胞凋亡的功能。结果获得1株可分泌抗DR5mAb的杂交瘤细胞系R150。SDS-PAGE电泳检测证实,获得的R150可特异性地识别DR5;R150的Ig亚类为IgGI(λ型);腹水效价为1×106;通过流式细胞仪可敏感地检测到肿瘤细胞表面DR5的表达水平及R150诱导肿瘤细胞凋亡情况。结论获得1株可分泌抗DR5mAb的细胞系R150,抗体具有效价高、特异性强等特点并能有效诱导肿瘤细胞凋亡,具有较好的应用价值。
Objective To prepare anti-human DR5 monoclonal antibody (mAb), identify its characteristics, and conduct biological activity analysis. Methods Balb / c mice were immunized with purified soluble sDR5 (sDR5) and hybridoma technology was used to prepare anti-human DR5 mAb. The binding properties of anti-DR5 mAb and sDR5 were determined by ELISA and SDS-PAGE electrophoresis. The anti-DR5 mAb The indirect ELISA was used to detect the titer of ascites mAb. Flow cytometry was used to detect the expression of DR5 on the surface of tumor cells. The function of anti-DR5 monoclonal antibody (mAb) on the apoptosis of tumor cells was detected by flow cytometry. As a result, a hybridoma cell line R150 secreting anti-DR5 mAb was obtained. The results of SDS-PAGE showed that R150 could specifically recognize DR5. The Ig subclass of R150 was IgGI (λ type); the titer of ascites was 1 × 106. The surface of tumor cells could be detected by flow cytometry DR5 expression and R150 induced tumor cell apoptosis. CONCLUSIONS: 1 R150 cell line secreting anti-DR5 mAb is obtained. The antibody has the characteristics of high titer and specificity, and can effectively induce apoptosis of tumor cells, which has a good application value.