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用人上皮癌细胞系A 431细胞作为抗原免疫BalB/c小鼠,制备七株抗人表皮生长因子受体的单克隆抗体的杂交瘤,这些杂交瘤经三次亚克隆后仍能稳定地分泌单克隆抗体。对其中四株杂交瘤分泌的单克隆抗体进行了鉴定。免疫沉淀放射自显影结果示单克隆抗体3、101和176均可识别A 431细胞膜抗原MW为170000的蛋白质即EGF受体。单克隆抗体59可以识别低分化鼻咽癌细胞膜上EGF受体。单抗3、176和59等可抑制EGF与受体的特异结合,而101和94则不能抑制EGF与受体的结合。 用Protein-A Sepharose CL4B纯化了单抗,纯化的单抗主要为IgG_1亚类。用SDS聚丙烯酰胺凝胶电泳对纯化的单抗进行了纯度测定。
Balb / c mice were immunized with human epithelial cancer cell line A 431 cells as an antigen to prepare seven hybridomas of monoclonal antibodies against human epidermal growth factor receptor. These hybridomas still stably secreted monoclonal after three subcloning antibody. Monoclonal antibodies secreted by four hybridomas were identified. The result of immunoprecipitation autoradiography showed that monoclonal antibodies 3, 101 and 176 both recognized the protein of A 431 cell membrane antigen MW 1700000 as EGF receptor. Monoclonal antibody 59 recognizes EGF receptors on poorly differentiated nasopharyngeal carcinoma cell membranes. McAbs 3,176 and 59 could inhibit the specific binding of EGF to the receptor, while 101 and 94 could not inhibit the binding of EGF to the receptor. Monoclonal antibodies were purified using Protein-A Sepharose CL4B and purified monoclonal antibodies were predominantly IgG1 subclasses. The purity of the purified mAb was determined by SDS polyacrylamide gel electrophoresis.