鼠抗人CD28分子单克隆抗体的研制及生物学特性研究

目的 制备鼠抗人CD28分子的单克隆抗体（mAb)，研究其在T细胞的活化、增殖及信号传导中的作用。方法 以小鼠淋巴瘤细胞转染人CD28基因的细胞株（CD28-T）为免疫原，采用B淋巴细胞杂交瘤技术，获取分泌特异性mAb的杂交瘤细胞株，以体内诱生法生产腹水，并以免疫亲和层析法对其纯化，以快速定性试纸法鉴定mAb的Ig亚类，竞争抑制法分析mAb识别的抗原表位，3H-TdR掺入法研究mAb对T细胞的刺激效应，结果 成功地获得了5株分泌特异性抗入CD28mAb的杂交瘤细胞株，鉴定的1株（克隆18G8）属IgG2a，腹水效价（流式细胞仪分析）达1：2400以上，该mAb能60%阻断标准鼠抗入CD28抗体与相应抗原的结合，提示其识别的抗原表位与标准mAb不完全相同，mAb18G8可取代B7-1分子介导的协同刺激信号，促进人外周血T细胞增殖（ST=7）。结论 18G8是12株功能性mAb，具有重要的研究和应用价值。

Preparation of functional monoclonal antibody against human CD28 and analysis of its biological feature

Aim To prepare the monoclonal antibodies (mAbs) against human CD28 and to study its biological feature. Methods The hybridoma cell lines were obtained by fusing spleen cells of Blab/c mice that had been immunized with murine lymphoma cells transfected with full-length huaman CD28 cDNA to myeloma cells Sp2/0. Ascites were induced to produce the mAbs. The specificity and affinity of the mAb 18G8 was verified by CD28 competitive inhibitory test and FACS. Reactivities of mAb 18G8 to PBTC, U266, 8226, Jurkat and Daudi cell were studied by indirect immunofluorescence staining. mAb 18G8-inducing proliferation of peripheral blood T cells (PBTCs) was determined by 3H]thymidine incorporation test. Results Five hybridoma cell lines were obtained. mAb 18G8 secreted by one of the them, belong to mouse IgG2a. It recognized a epitope different from which recognized by the standard mAb(clone CD28.2). The Reactivitrates of the mAb 18G8 to PBTC, U266, 8266, Jurkat and Daudi cells were 70.2％ , 99.3％ , 98.6％ , 76.4％ and 1.9％ , respectively, similar with CD28.2. It was indicated that different antigen epitopes expressed on all above cells. mAb 18G8 could promote the PBTC proliferation in vitro(SI=7). It was indicated that The substitution of mAb 18G8 for B7-1 molecule could also mediate the costimulatory signals. Conclusion 18G8 is a specific and functional anti-CD28 mAb it may be of significant value in basic studies and clinical application.