重组人源化兔抗vEGF单克隆抗体的抗原结合活性测定_577.doc

重组人源化抗抗体的抗原结合活性测定【摘要】目的 采用直接ELISA法检测人源化抗抗体的抗原结合。方法 以固定量的做包被抗原，辣根过氧化酶标记的抗人IgG（H+L）为二抗，直接测定不同浓度参考品和供试品与抗原的结合活性，通过数据处理软件对结果绘图，根据供试品及参考品EC50值计算供试品的相对结合力。结果同一批次的品分别经3次测定，相对结合分别为（±18.74）%、（±18.4）% 和（10±18.47）%，平均值（±16.57）。3次试验的变异系数分别为2%、14.%和%。结论 直接ELISA法精密性良好，结果客观，影响因素少，可用于抗原结合活性的常规检测。 【关键词】；相对抗原结合力；直接ELISA(通讯作者：饶春明010 Email: raocm@nicpbp.org.cn Measurement of the Antigen Binding Activity of the Humanized Anti-vEGF Rabbit Monoclonal Antibody Fan Wenhong, Bi Hua, Rao Chunming （National Institute for the Control of Pharmaceutical and Biological Products of China, Beijing 100050） 【Abstract】 Objective To determine the antigen binding activity of the humanized antibody by direct ELISA. Methods The reference and the samples of humanized anti-vEGF rabbit monoclonal antibody were serially diluted and allowed to incubate with a fixed amount of recombinant vEGF-Fc. The EC50 of reference standard and the samples would be determined by fitting a 4-parameter equation. The binding potency of the samples would be expressed as a percentage of the EC50 to that of the reference standard. Results The average binding potency of the humanized rabbit monoclonal antibody bulk was (106.94±16.57)%，from 3 times tests of vial samples of the same batch，which quality control. The results were (90.43±18.74)%, (123.56±18.4)% and (106.83±18.47)% respectively. The relative binding potency of the final products were (97.34(18.42)%, (123.30(11.08)% and (93.91(4.57)%. The CVs of three tests are 18.93%, 8.96% and 4.87% respectively. Conclusions The results showed that direct ELISA is a repeatable and stable method to detect the relative binding affinity. The method could be used as a routine analysis in the quality control of humanized anti-vEGF rabbit monoclonal antibody. 【Key words】; relative binding potency; direct ELISA 血管内皮生长因子（Vascular Endothelial Growth Factor，EGF）是由某些肿瘤细胞分泌通过与血管内皮上的相应受体结合促进内皮细胞增殖，同时可增加血管通透性使内皮细胞迁移，诱导肿瘤血管生成，维持肿瘤的继续生长，是目前发现的最强烈的血管生成因子，与诸多生理及病理过程有关。[4,5]。肿瘤抗血管生成治疗理论在上世纪70