【摘要】 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。更多还原

【Abstract】 AIM:To prepare mouse anti-human PD-1 monoclonal antibodies (mAbs) and identify their biological characteristics. METHODS:The BALB/c mice were immunized with the transfected cell line PD-1/L929. The cells were fused with Sp2/0 using monoclonal antibody techniques and the positive clones were screened by FCS with PD-1/L929. The secreted anti-PD-1 mAbs were identified through rapid isotyping analysis,karyotype analysis,Western blot,competitive inhibition test,indirect immunofluorescence assay,and tumor cell lines detection. RESULTS:Two mouse anti-human PD-1 hybridomas were obtained and their secreted mAbs were named (1F2 and 5F10). Their biological characteristics suggested that they could recognize a protein with approximate molecular weight 55 000 on PD-1/L929 cell lines and different epitopes. 1F2 could recognize PD-1 molecules expressed on SKHep-1 and 7721 while 5F10 could recognize Raji cells. CONCLUSION:Two mouse anti-human PD-1 hybridoma cell lines and their secreted monoclonal antibodies have been successfully obtained and identified.更多还原