犬PD-1单克隆抗体的筛选与鉴定

Development and Characterization of Mouse Anti-canine PD-1 Monoclonal Antibodies

关键词:  犬  PD-1  单克隆抗体  肿瘤

Key words:  cannie  PD-1  monoclonal antibody  cancer

摘要:

为筛选犬程序性死亡受体1(cPD-1)单克隆抗体，以原核表达并纯化复性的cPD-1胞外区蛋白为靶抗原免疫BALB/c小鼠，应用单克隆抗体杂交瘤细胞技术和间接ELISA方法，经3轮轮筛选和克隆化，获得1株能稳定分泌抗cPD-1单克隆抗体的杂交瘤细胞株1F9。间接ELISA检测显示1F9杂交瘤细胞株连续传代培养能稳定分泌单克隆抗体，效价达到1∶2048，亚型鉴定重链为IgG1，轻链为κ；以HEK293细胞表达cPD-1胞外区蛋白为检测抗原，Western-blot和间接免疫荧光试验进行鉴定，结果显示1F9单克隆抗体能特异性结合cPD-1胞外区蛋白。本研究通过小鼠杂交瘤技术成功筛选到1株鼠源cPD-1单克隆抗体。

Abstract:

In order to screen the monoclonal antibody against canine programmed death receptor 1 (cPD-1),BALB/c mice were immunized with prokaryotically expressed and purified recombinant cPD-1 extracellular region protein as the target antigen, and a hybridoma cell line 1F9, which could stably secrete anti-cPD-1 monoclonal antibody, was obtained after 3 rounds of screening and clonation by applying monoclonal antibody hybridoma cell technology and indirect ELISA method. Indirect ELISA showed that the 1F9 hybridoma cell line was able to stably secrete monoclonal antibodies with a potency of 1∶2048 in continuous passaging culture, and the heavy chain was identified as IgG1 and the light chain as κ by subtype identification; the cPD-1 extracellular region protein expressed by HEK293 cells was used as the detection antigen, and Western-blot and indirect immunofluorescence assays were used for identification; the results showed that 1F9 monoclonal antibody could specifically bind to cPD-1 extracellular region protein. In this study, a mouse-derived cPD-1 monoclonal antibody was successfully screened by mouse hybridoma technique.