摘要
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。
A 431 cells have been used as an immunogen for development of monoclonal antibodies against the epidermal growth factor receptor (EGF-R) . The A 431 antigen recognized by MoAb 3, 176 and 101 has an apparent molecular weight of approximately 170 000. It is a cell surface protein that can bind to above MoAbs in a number of human cell lines to a degree which parallels ECF binding. MoAb 59 was able to recognize the same molecular weight protein on the CNE-2 cell (poorly differentiated nasopharyngeal carcinoma) as on the A 431 cell. It was shown that MoAb 3, 176 and 59 inhibited EGF binding while MoAb 101 didn't. We therefore conclude that MoAb 3, 176 and 59 are directed against binding site on the human EGF receptor.Purification of monoclonal antibody was carried out by protein-A sepharose CL4B. Purity of antibody and isoelectric focusing immunoglobulins were estimated.