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目的:制备抗人DcR3单克隆抗体(mAb),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DcR3mAb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DcR3mAb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(1B1)可与SW480细胞成分反应。结论:成功建立稳定分泌抗人DcR3mAb的杂交瘤细胞株,其分泌的抗体特异性强、效价高,为研究DcR3在组织中的表达、分布及研制ELISA试剂盒奠定基础。
Objective: To prepare anti-human DcR3 monoclonal antibody (mAb) and identify its specificity. Methods: The purified His-DcR3 fusion protein was used to immunize mice. The anti-human DcR3 mAb was purified by using lymphocyte hybridoma technique. The purified antibody was identified by Western blot and ELISA. Results: Five hybridoma cells secreting stable anti-DcR3 mAb were all IgG1 subtype. The antibody titer of ascites was 1 × 10-5 ~ 1 × 10-7. Western blot showed that mAb secreted by 5 cells could be identified DcR3 protein, one of which (1B1) can react with SW480 cell components. CONCLUSION: The hybridoma cell line secreting anti-human DcR3 mAb was successfully established and its secreted antibody was highly specific and high in potency. It laid the foundation for the study of the expression and distribution of DcR3 in tissue and the development of ELISA kit.