鼠抗人B7-2单克隆抗体的研制及生物学特性研究

Preparation of functional monoclonal antibody against human B7-2 molecule and analysis of its biological characteries

以天然高表达膜型B7-2分子的人恶性B淋巴瘤经胞株Daudi和B7-2基因转染细胞株L929-B7-2为免疫原及检测细胞株,采用B细胞杂交瘤技术建立了1株稳定分泌抗人B7-2单抗的杂交瘤,命名为6A5。快速定性试纸法鉴定6A5属小鼠IgG1亚类。经体内诱生腹水法制备单抗,小鼠腹水形成的阳性率为80%,腹水的收获量平均为5.9 ml/只小鼠。经免疫亲和层析法纯化,腹水中抗体蛋白的得率为1.1 mg/ml。采用间接免疫荧光法及流式细胞仪分析,单抗6A5与L929-B7-2、PBTC、Raji和Daudi的阳性结合率分别为99.9%、4.6%、90.6%和99.1%。将6A5(终浓度为5μg/ml)加入到Daudi细胞的培养体系中,经显微镜观察及MTT法分析,6A5对Daudi细胞的生长在24 h内具有抑制作用(P<0.05)。以丝裂霉素处理的L929-B7-2为刺激细胞,PBTC为反应细胞,加入6A5共同培养。经MTT法分析,6A5能阻断B7-2介导的协同刺激信号,抑制PBTC增殖(P<0.05)。提示6A5是一株功能性抗体,在B7-2分子相关的基础和应用研究中具有重要的价值。

The cell lines 1.929-B7-2, transfected human B7-2 gene, and Daudi, established from human B cell malignant lymphoma patient were taken as immunogen, pretreated with mitomycin, before being used to immunize BALB/c mice, and the splenocytes of immunized mice were used to fuse with murine myeloma cells SP2/0. One hybridoma, named 6A5, stably secreting monoclonal antihody against human B7-2 molecule was successfully obtained after repeated secreting and sub cloning culture. The subclass of 6A5 was mouse IgG1 with kappa light chains through fast-strip method analysis. Ascites were induced to produce monoclonal antibody and 5.9 ml ascites was obtained from each BAI.B/c mouse. The purified 6A5 from ascites was about 1.1 mg per ml ascites with affinity chromatography method. The 6A5 could specifically recognize membrane B7 2 molecules expressed on cells of 1.929-B7-2, PBTC, Raji and Daudi by indirect immunofluoresence and the positive percentage was 99.9%, 4.6%, 90.6%and 99.1%, respectively. In addition it could inhibit the growth and proliferation of Daudi cells and PBTC through blockade the B7-2 costimulatory signals with MTT assay. These results demonstrate that 6A5 is a funtionally specific monoclonal antibody for B7-2 molecule and has a high affinity for its ligands. It may be of significant value in basic studies and clinical applications.