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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。
Objective : Cell surface epitope engraftment (SEM) is a new way to select specific expression molecules on the cell surface to prepare monoclonal antibodies. Since P185 neu c erbB 2 is an important surface marker of breast cancer and other tumors, this study aims to use the SEM method to prepare P185 monoclonal antibody with good specificity and high sensitivity and analyze its characteristics for clinical application. METHODS: BALB C mice were immunized with the cell surface epitope-encapsulation method, cell fusion was performed with the conventional hybridoma technique, ELISA assay, and cloning with the limiting dilution method. RESULTS : Three McAb hybridoma cells that stably secreted the anti-cancer gene erbB 2 product P185 were obtained. Indirect immunoperoxidase staining and flow cytometric analysis demonstrated that this McAb specifically binds to P185 positive cell membranes but not to structurally similar EGFR membrane positive cells. Western blotting experiments demonstrated that these McAbs specifically bind to P185 protein, and immunohistochemistry results of pathological sections demonstrated that the monoclonal antibody showed specific membrane-positive responses to breast cancer. Conclusion: The McAb hybridoma of tumor surface antigen P185 prepared by SEM method has a very good value for clinical diagnosis of tumor antigen P185 and its prospect of engineering transformation.