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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 B7 cell hybridoma was used as the immunogen and cell line L929-B7-2, which was transfected by Daudi and B7-2 gene of human malignant B lymphoma with high expression of membrane type B7-2 molecule. A stable hybridoma secreting anti-human B7-2 monoclonal antibody was named 6A5. 6A5 is a mouse IgG1 subclass identified by rapid qualitative test paper method. The in vivo induced ascites method of preparation of monoclonal antibody, the formation of ascites in mice the positive rate was 80%, the average amount of ascites harvest of 5.9 ml / mouse. Purified by immunoaffinity chromatography, the yield of antibody protein in ascites was 1.1 mg / ml. The positive binding rates of monoclonal antibody 6A5 to L929-B7-2, PBTC, Raji and Daudi were 99.9%, 4.6%, 90.6% and 99.1% respectively by indirect immunofluorescence and flow cytometry analysis. 6A5 (final concentration 5μg / ml) was added to the culture system of Daudi cells. The results of MTT assay showed that 6A5 could inhibit the growth of Daudi cells within 24 hours (P <0.05). Mitomycin-treated L929-B7-2 was stimulated with PBTC as a reactive cell and plated in 6A5. By MTT assay, 6A5 blocked the B7-2-mediated costimulatory signal and inhibited PBTC proliferation (P <0.05). Tip 6A5 is a functional antibody, B7-2 molecules related to the basic and applied research has important value.