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鼠抗人OX40分子激发型单克隆抗体(mAb)的研制及其生物学特性的鉴定。以高表达人OX40分子的基因转染细胞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,将小鼠脾脏细胞与骨髓瘤细胞SP2/0融合,并以L929-OX40细胞为阳性筛选细胞,L929-Mock细胞为阴性筛选细胞,采用免疫荧光技术对杂交瘤进行反复筛选及多次克隆化培养;采用快速定性试纸法检测抗体亚型;竞争抑制实验分析抗原结合表位;Western blot检测抗体特异性;采用MTT法分析单抗在体外对T细胞增殖的促进作用,并采用ELISA检测培养上清中的细胞因子。结果:获得一株稳定、高效分泌鼠抗人OX40单克隆抗体的杂交瘤细胞株(克隆号:9H8),该株mAb能够特异性识别U937、Jurkat、Thp-1等血液来源肿瘤细胞株表面OX40分子,并且能够有效促进T细胞活化、增殖及细胞因子的分泌。成功研制了一株分泌鼠抗人OX40分子激发型单克隆抗体的杂交瘤细胞株,该抗体能够特异性识别人OX40分子并能够介导OX40正性协同刺激效应。
Development of a Murine Antihuman OX40 Molecularly Induced Monoclonal Antibody (mAb) and Identification of Its Biological Characteristics. The BALB / c mice of 6-8 weeks old were routinely immunized with gene-transfected L929-OX40, a gene overexpressing human OX40, and the spleen cells of mice were immunized with B lymphocyte fusion technique / 0 fusion, L929-OX40 cells positive cells were screened, L929-Mock cells were negative screening cells, immunofluorescence technique hybridomas were repeatedly screened and multiple clonal culture; rapid detection of antibody subtypes The competitive inhibition assay was used to analyze the antigen-binding epitope. The specificity of the antibody was detected by Western blot. The promotion of T cell proliferation by monoclonal antibody was analyzed by MTT assay. The cytokines in the culture supernatant were detected by ELISA. Results: A stable and highly efficient hybridoma cell line secreting mouse anti-human OX40 monoclonal antibody (clone number: 9H8) was obtained. The mAb could specifically recognize the surface OX40 of U937, Jurkat, Thp-1 and other blood-derived tumor cell lines Molecules, and can effectively promote T cell activation, proliferation and secretion of cytokines. We successfully developed a hybridoma cell line secreting murine anti-human OX40 molecularly excited monoclonal antibody that specifically recognizes human OX40 molecule and mediates OX40 positive costimulation effects.