抗人肾癌G250单克隆抗体的制备及其功能鉴定

目的 大量制备、纯化抗人G250单克隆抗体(G250-MeAb),并鉴定其与G250合成多肽及细胞天然抗原的结合特异性.方法 将抗人G250杂交瘤细胞接种BALB/C小鼠腹腔,大量制备G250-McAb.采用正辛酸-硫酸铵沉淀法获得粗制抗体,然后经蛋白A亲和层析柱纯化抗体.采用荧光激活细胞分类术及免疫组织化学法鉴定G250-McAb与抗原结合特异性.结果 成功制备抗人G250-MeAb,腹水经纯化后,获得了高纯度抗人G250单克隆抗体,蛋白浓度达4.78 g/L.荧光激活细胞分类术及免疫组织化学结果 显示G250-McAb与G250表达阳性的Ketr-3细胞特异性结合,而不与G250表达阴性的ACHN细胞结合.结论 成功制备并纯化G250-McAb,为进一步开展G250-McAb介导的肾癌诊断、治疗奠定了基础.

Preparation and functional identification of anti-human-G250 monoclonal antibody

Objective To prepare and purify anti-human-G250 monoclonal antibody (G250-McAb) and identify its function. Methods G250-McAb was generated in BALB/C mice abdominal cavity by injecting intraperitoneally with 1 × 106 hybridoma cells. The purity of McAb was determined by ammonium sulphate. The specificity of the McAb with G250 antigen was identified by fluorescence-activated cell sorting (FACS) and immunohischemistry. Results The McAb with high purity was obtained and the final concentration of McAb protein was about 4.78 g/L. It was also verified by FACS and immunohischemistry that McAb could bind to the surface of the G250 antigen positively expressed cells such as Ketr-3 but not to the G250 antigen negatively expressed cells such as ACHN. Conclusion Anti-human-G250 McAb with high specificity was successfully generated and evaluated,which can be used in the diagnosis and treatment of renal cancer.