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目的采用直接ELISA法测定重组人源化兔抗血管内皮生长因子(Vascular endothelial growth factor,VEGF)单克隆抗体的抗原结合活性。方法以VEGF-Fc为包被抗原,采用直接ELISA法测定不同浓度重组人源化兔抗vEGF单抗参考品和供试品与抗原的结合活性,根据供试品及参考品的EC50值计算供试品的相对结合力,并分析该法的精密性。结果重组人源化兔抗VEGF单抗供试品及参考品的抗原结合均存在量效关系,且符合四参数方程。同一批次的供试品单抗原液的相对结合活性分别为参考品的(90.43±18.74)%、(123.56±18.40)%和(106.83±18.47)%,均值为(106.94±16.57)%。3次试验的变异系数分别为20.72%、14.89%和17.29%。同一批次的供试品成品的相对结合活性分别为(97.34±18.42)%、(123.30±11.08)%和(93.91±4.57)%,变异系数分别为18.93%、8.96%和4.87%。结论 直接ELISA法精密性良好,操作简便,可用于重组人源化兔抗VEGF单抗抗原结合活性的常规检测。
Objective To determine the antigen-binding activity of recombinant humanized rabbit anti-vascular endothelial growth factor (VEGF) monoclonal antibody by direct ELISA. Methods The VEGF-Fc-coated antigen was used to measure the binding affinity of the recombinant humanized rabbit anti-vEGF monoclonal antibody (mAb) with the antigen and its binding activity to the test sample by the direct ELISA method The relative strength of the test sample, and analyze the precision of the law. Results The results showed that there was dose-response relationship between the recombinant humanized rabbit anti-VEGF monoclonal antibody and the reference product, and the four-parameter equation was followed. The relative binding activities of the same batch of test single antigen solution were (90.43 ± 18.74)%, (123.56 ± 18.40)% and (106.83 ± 18.47)%, respectively, with the mean of (106.94 ± 16.57)%. The coefficient of variation of three experiments were 20.72%, 14.89% and 17.29% respectively. The relative binding activities of the same batch of finished products were (97.34 ± 18.42)%, (123.30 ± 11.08)% and (93.91 ± 4.57)%, respectively, with coefficients of variation of 18.93%, 8.96% and 4.87%, respectively. Conclusion The direct ELISA method has good precision and simple operation, and can be used for the routine detection of recombinant humanized anti-VEGF mAb antigen binding activity.