鼠抗人OX40L功能性单克隆抗体的研制及其生物学特性的鉴定

目的　鼠抗人OX4 0L分子功能性单克隆抗体的研制及其生物学特性的鉴定。方法以高表达人OX4 0L分子的转基因细胞L92 9/OX4 0L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 ,并以L92 9/OX4 0L为阳性抗体筛选细胞 ,L92 9/mock为阴性抗体筛选细胞 ,经免疫荧光标记和流式细胞术分析、反复筛选和多次克隆化培养 ,筛选出特异分泌鼠抗人OX4 0L分子单克隆抗体的杂交瘤细胞株 ;采用Westernblot、Ig亚型快速定性试纸法、间接免疫荧光法、竞争结合抑制试验和3 H TdR增殖试验等对单抗进行生物学特性的鉴定。结果　成功获得 3株持续、稳定分泌鼠抗人OX4 0L单克隆抗体的杂交瘤细胞株 ,命名为 9H10、4C12和 1G1。对单抗的生物学功能的研究结果表明 ,3株单抗均能识别活化B细胞和成熟DC表达的OX4 0L分子 ,且能抑制成熟DC对T细胞的促增殖作用 ,并与阻断型抗人B7 1单抗具有协同作用。结论　获得的 3株分泌鼠抗人OX4 0L功能性单克隆抗体的杂交瘤 ,所分泌的抗体具有特异性地识别人OX4 0L分子并能阻断OX4 0 /OX4 0L共刺激信号及抑制DC对T细胞的激发作用。

Preparation of anti-human OX40L functional monoclonal antibody and characterization of its biological activities

Objective To prepare an anti-human OX40L functional monoclonal antibody and to characterize its biological activities. Methods A stable human OX40L transfected cell line L929/OX40L was used as an antigen to immunize BALB/c mice. By means of the cell fusion hybridoma technique and multiple cell subcloning and repeated screening with L929/OX40L as the antibody screening positive cell while L929/mock as the negative control, the hybridoma cell lines specifically secreting anti-OX40L monoclonal antibodies were selected. Then their biological activities were investigated by Western blot, fast-strip murine Ig subclass typing method, indirect immunofluorescence, competitive inhibition test and 3H-TdR incorporation assay. Results Three hybridoma cell lines, 9H10, 4C12 and 1G1, with the property of secreting specific anti-OX40L monoclonal antibody continuously and steadily were successfully obtained. Their biological activity study showed that these monoclonal antibodies could bound to human OX40L epitopes on activated B cells and maturate DCs and inhibit the proliferation of T lymphocytes costimulated by DCs. Furthermore, three anti-OX40L monoclonal antibodies also synergize with the anti-B7-1 monoclonal antibody to block the DC-inducing T cell proliferation in vitro. Conclusion Three hybridoma cell lines which secrete anti-OX40L monoclonal antibodies continuously and steadily have been established. These monoclonal antibodies could specifically recognize OX40L molecule and block OX40/OX40L costimulatory signals to suppress the proliferation of T lymphocytes costimulated by DCs.