鼠抗人PD-L1单克隆抗体的制备及鉴定

Preparation and Identification of a Monoclonal Antibody Against Human PD-L1

目的制备鼠抗人PD-L1单克隆抗体(mAb)并对其生物学特性进行鉴定。方法以重组GST-PDL1融合蛋白免疫BALB/c小鼠,建立可稳定分泌抗PD-L1mAb的杂交瘤细胞株。用ELISA及Western blot鉴定mAb的特性,并测定其效价及Ig亚类。用免疫组织化学和流式细胞术分析其与干扰素-γ(IFN-γ)诱导的MDA-MB-231细胞表面PD-L1的结合能力。用CFSE标记外周血单个核细胞,通过流式细胞分析抗PD-L1单抗对抗原诱导的T细胞活化、增殖的影响。结果获得了一株可稳定分泌鼠抗人PD-L1mAb的杂交瘤细胞株。杂交瘤细胞培养上清效价为1:6400,腹水效价为1:102400,Ig亚类为IgG1。Western blot显示其能特异性结合重组GST-PDL1抗原。能与IFN-γ诱导表达的肿瘤细胞表面PD-L1结合,能封闭PD-1/PD-L1通路,促进抗原诱导的T细胞增殖反应。结论成功获得了具有高活性鼠抗人PD-L1杂交瘤及其分泌的mAb。

Objective To prepare mouse anti-human PD-L1 monoclonal antibodies（mAbs） and identify their biological characteristics.Methods BALB/c mice were immunized with recombinant GST-PDL1 protein,and the strain of hybridoma cell secreting anti-PD-L1 mAb was obtained.The specificity of anti-PD-L1 mAb was checked and evaluated with ELISA and Western blot.Its titer,immunoglobulin subtype were also measured.The combining capacity of anti-PD-L1 mAb with PD-L1 on MDA-MB-231 cells,which had been induced with IFN-γ for 72 hours,was identified through immunohistochemistry and flow cytometry.The peripheral blood mononuclear cell was labled with CFSE,and the effect of anti-PD-L1 mAb on the activation and proliferation of the lymphocyte induced with antigens was evaluated by flow cytometry analysis.Results A strain of hybridoma cell secreting anti-PD-L1 mAb was successfully obtained and identified belong to IgG1 subtype.Its titers in cultural supernatant and ascetic fluid were 1：6400 and 1：102400,respectively,by ELISA.The purified mAbs showed good affinity and specificity against GST-PDL1 protein in Western blot,also could block the PD-1/PD-L1 pathway and promote the proliferation of lymphocyte induced with antigens.Conclusion Hyperactivity mouse anti-human PD-L1 hybridoma cell lines and their secreted monoclonal antibodies have been successfully obtained and identified.