表皮生长因子受体Ⅲ型突变体(EGFRvⅢ)单链抗体亲和力检测的间接ELISA的建立及优化

Establishment and optimization of an ELISA for affinity detection of single-chain antibodies to EGFRvⅢ

目的建立检测表皮生长因子受体变体Ⅲ(EGFRvⅢ)单链抗体PD0721亲和力检测的ELISA,并且对实验条件进行优化。方法利用本课题组制备的单链抗体PD0721,建立检测PD0721单链抗体亲和力的间接ELISA,并采用棋盘滴定法对实验条件中的抗体抗原浓度、二抗浓度、包被条件等方面进行优化,对本方法的灵敏度及精密度进行考察。结果使用PBS稀释抗原至1.25 mg/L于4℃包被12 h,加入120 ng/mL PD0721单链抗体以及1∶8000的酶标抗体为最佳检测结果。优化后的结果,在PD0721单链抗体浓度为15 ng/mL~480 ng/mL范围内回归模型拟合效果良好,最低检测限为7.5 ng/mL。组内差异系数为0.11%~0.99%,组间差异系数为0.68%~3.15%。结论建立了能准确、稳定检测PD0721单链抗体亲和力的间接ELISA。

Objective To establish an ELISA for the detection of the affinity of the epidermal growth factor receptor variant Ⅲ(EGFRvⅢ) single-chain antibodies PD0721, and optimize the experimental conditions. Methods An indirect ELISA for the detection of the affinity of the PD0721 single-chain antibody which was prepared by our laboratory was established. By square matrix titration method of indirect ELISA, the experimental conditions including antibody and antigen concentrations and coating conditions were optimized, and finally the sensitivity and precision of the method were analyzed. Results The antigen was diluted with PBS to 1.25 mg/L and coated at 4 ℃ for 12 hours, 120 ng/mL PD0721 single-chain antibody and enzyme-labeled antibody at the dilution of 1∶8000 were added for the best results. Indirect ELISA exhibited high performance within a dynamic range 15 ng/mL-480 ng/mL, and the limit of detection(LOD) was 7.5 ng/mL. The intra-assay coefficient variations(CV) ranged from 0.11% to 0.99% and inter-assay CV ranged from 0.68% to 3.15%. Conclusion An accurate and stable ELISA for detecting the affinity of PD0721 single-chain antibody has been established, which laid a foundation for future preparation of antibody-conjugated drugs.