【摘要】 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。更多还原

【Abstract】 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.更多还原