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目的:研制功能性鼠抗人OX40单克隆抗体。方法:以转人OX40的转基因细胞L929-OX40为免疫原,常规免疫6-8周龄的雌性BALB/c小鼠;采用B淋巴细胞融合技术,将免疫小鼠脾脏细胞与SP2/0融合,以L929-OX40转基因细胞及PHA活化的T细胞为抗体筛选阳性细胞,经免疫荧光标记分析对杂交瘤进行反复筛选和多次的克隆化培养;采用快速定性试纸法及竞争抑制结合试验分析了该单抗的亚类及抗原识别位点;采用MTT法分析单抗在体外对T细胞的促增殖效应以及ELISA分析活化T细胞分泌的细胞因子。结果:获得1株持续、稳定分泌鼠抗人OX40单克隆抗体的杂交瘤细胞株(命名为7E11),该单抗能特异性地识别人OX40分子和介导有效的共刺激信号,体外促进活化的T细胞增殖和细胞因子的分泌。结论:成功研制成一株能分泌功能性鼠抗人OX40单克隆抗体的杂交瘤,该抗体特异性地识别人OX40分子并具有在体外协同刺激T细胞的作用。
Objective: To develop a functional mouse anti-human OX40 monoclonal antibody. METHODS: Female BALB / c mice of 6-8 weeks old were routinely immunized with L929-OX40 transgenic OX40 transgenic mice. The spleen cells of immunized mice were fused with SP2 / 0 using B lymphocyte fusion technique. Positive cells were screened by L929-OX40 transgenic cells and PHA-activated T cells. The hybridomas were screened and cloned repeatedly by immunofluorescent labeling analysis. The rapid qualitative test strips and competitive inhibition assay Mabs subclasses and antigen recognition sites. MTT assay was used to analyze the proliferative effect of mAb on T cells in vitro and the cytokines secreted by activated T cells by ELISA. Results: One hybridoma cell strain (named as 7E11) which secreted a stable and stable murine anti-human OX40 monoclonal antibody was obtained. The monoclonal antibody specifically recognizes human OX40 molecule and mediates potent co-stimulatory signals and promotes activation in vitro T cell proliferation and cytokine secretion. CONCLUSION: A hybridoma secreting a functional mouse anti-human OX40 monoclonal antibody has been successfully developed, which specifically recognizes human OX40 molecule and has the synergistic effect of stimulating T cells in vitro.