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目的:制备鼠抗人PD-1单克隆抗体(mAb)及鉴定其生物学特性。方法:以基因转染细胞PD-1/L929为免疫原,免疫BALB/c小鼠;采用淋巴细胞杂交瘤技术进行细胞融合,经选择性克隆化培养及流式细胞术(FCM)分析,筛选分泌鼠抗人PD-1分子mAb的杂交瘤细胞株;采用Ig亚型快速定性试纸法、染色体核型分析、Western blot、竞争结合抑制试验及肿瘤细胞株测定等方法对mAb进行生物学特性鉴定。结果:获得了2株持续稳定分泌鼠抗人PD-1mAb的杂交瘤细胞株,命名为1F2和5F10。对其生物学特性的鉴定结果表明,均为条带相对分子质量在(Mr)55000左右的免疫球蛋白,具有不同的PD-1结合位点;1F2mAb可识别SKHep-1和7721细胞株表面的PD-1分子,而5F10可识别Raji细胞株的PD-1分子。结论:成功获得了2株鼠抗人PD-1杂交瘤及其分泌的mAb。
Objective: To prepare mouse anti-human PD-1 monoclonal antibody (mAb) and to identify its biological characteristics. Methods: BALB / c mice were immunized with PD-1 / L929 cells. The cells were fused by lymphocyte hybridoma technique. The cells were selected by clonal culture and flow cytometry (FCM) The hybridoma cell lines secreting murine anti-human PD-1 molecule mAb were used to characterize the biological characteristics of mAbs by rapid qualitative test of Ig subtype, karyotype analysis, Western blot, competitive inhibition test and tumor cell line assay . Results: Two hybridoma cell lines stably secreting mouse anti-human PD-1 mAb were obtained and named as 1F2 and 5F10. The results of their biological characteristics showed that all of them were immunoglobulins with a molecular weight of about 55000 and a different PD-1 binding site. 1F2mAb recognized the surface of SKHep-1 and 7721 cell lines PD-1 molecule, while 5F10 recognizes the PD-1 molecule of Raji cell line. Conclusion: Two murine anti-human PD-1 hybridomas and their secreted mAbs were successfully obtained.