摘要
目的:研制特异性鼠抗人PD-L1单克隆抗体(mAb)并鉴定其生物学特性。方法:以高表达人PD-L1分子的基因转染细胞L929/PD-L1为免疫原,常规免疫BALB/c小鼠,采用B淋巴细胞杂交瘤技术进行细胞融合,以L929/PD-L1细胞为抗体筛选细胞,L929/mock细胞为对照细胞,经选择性克隆化培养及流式细胞术(FCM)分析,筛选稳定和持久分泌鼠抗人PD-L1 mAb的杂交瘤细胞株;采用Ig亚型快速定性试纸法、Western blot、间接免疫荧光法和竞争结合抑制实验等方法对mAb进行生物学特性鉴定;继而体外实验分析mAb对T细胞增殖的影响。结果:成功获得3株鼠抗人PD-L1mAb,分别命名为11G8,2G5和5C3;对其生物学功能的研究结果显示,3株mAb均能识别活化T细胞表面PD-L1分子,和在体外显著促进T细胞的增殖。结论:获得的3株鼠抗人PD-L1功能性mAb,通过阻断PD-1/PD-L1抑制途径能够有效增强T细胞体外增殖,这为进一步研究PD-1/PD-L1信号通路提供了物质基础。
AIM： To prepare a functional mouse antihuman PD-L1 monoclonal antibody and to characterize its biological activities. METHODS： A stable human PD-L1 transfected cell line L929/PD-L1 was used as an antigen to immunize BALB/c mice. By means of the cell fusion technique, multiple cell subcloning, repeated screening with L92,9/ PD-L1 as target cells and the L929/mock cells used as the negative control, the hybridomas specifically secreting mouse anti-PD-L1 monoclonal antibodies were generated. Then its biological characterization was investigated by rapid murine Ig-subclass typing, Western blotting, indirect immune of luorescene assay, mutual competitive inhibition test. By means of MTT incorporation assay, detected the infection of mAb to T cell proliferation. Three mouse anti-human PD-L1 monoclonal antibodies were generated, named as llGS, 2G5 and 5C3. RESULTS： The results of characterization study showed that the monoclonal antibodies could recognize the PD-L1 on the activiated T cells. The mAbs could promote T cells proliferation. CONCLUSION： It is evident that the functional monoclonal antibodies for human PD-L1 have been generated, and it would provide the initial material for further study on the role of PD-1/PD-L1 signaling pathways.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2011年第11期1208-1211,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学青年基金资助(30800997)
