表面表位包埋法制备肿瘤表面抗原P185neu/c-erbB-2单克隆抗体及其性质研究

目的 :细胞表面表位包埋法 (SEM)是一种选择细胞表面特定的表达分子制备单克隆抗体的新途径。由于P185 neu c erbB 2 是乳腺癌及其他肿瘤的一个重要表面标志 ,这个研究旨在应用SEM法制备特异性好 ,灵敏度高的P185单克隆抗体并分析其特性以便于临床。方法 :采用细胞表面表位包埋法免疫BALB C小鼠 ,常规杂交瘤技术进行细胞融合 ,ELISA法测定 ,有限稀释法克隆化。结果 :筛选得到 3株能稳定分泌抗人癌基因erbB 2产物P185蛋白的McAb杂交瘤细胞。经间接免疫过氧化物酶染色和流式细胞术分析证明该McAb特异性与P185阳性细胞膜结合 ,而不与结构类似的EGFR膜阳性细胞结合。免疫印迹实验证明这些McAb特异地与P185蛋白结合 ,病理切片免疫组化结果证明该单抗对乳腺癌组织呈特异膜阳性反应。结论 :SEM法制备的肿瘤表面抗原P185的McAb杂交瘤具有很好的用于临床诊断肿瘤抗原P185的价值及工程化改造的前景。

Generation and characterization of monoclonal antibodies specific for the oncogene product P185neu/c-erbB-2 by surface epitope masking(SEM)

Objective:Surface epitope masking(SEM) is a new approach for selective production of monoclonal angibodies reacting with defined cell surface expressed molecules. Since P185 neu/c-erbB-2 protein is an important surface marker of breast cancer and other cancers This study was to prepare and characterize McAb against P185 neu/c-erbB-2 with high specificity and sensitivity by SEM for clinical application Methods:Using the method of Surface epitope masking(SEM)immunized BALB/C mouse with T6-17 cells which are NIH/3T3 transfected with meu/c-erbB-2 oncogene Fused the cells with the standard hybridma technique Performed ELISA screen to test the hybridomas'quality,limited dilution for cell cloning Results:Three hybridomas which can steadly secret McAb against P185 neu/c-erbB-2 protein were obtained The indirect immuno-HRP staining,flow cytometry analysis indicated that these three McAbs bind specifically to the P185 membrane positive cells,but not EGFR membrane positive cells Western blotting indicated that they all bind specifically to P185 protein Immunohistochemistry test showed positive staining for the sections of breast cancer tissue specifically Conclusion:These three McAbs are highly valued for clinic diagnosis and engineering