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

作　　者：张煜彬 吴忠秀 何彬[3] 杨畅[1] 李勇军[3] 刘亭[1] ZHANG Yubin;WU Zhongxiu;HE Bin;YANG Chang;LI Yongjun;LIU Ting(Guizhou Provincial Key Laboratory of Pharmaceutics/State Key Laboratory of Functions and Applications of Medicinal Plants,Guizhou Medical University,Guiyang 550004;School of Pharmacy,Guizhou Medical University,Guiyang 550025;Ministry-of-Education Engineering Research Center for the Development and Application of Ethnic Medicine and TCM,Guizhou Medical University,Guiyang 550004,China)

机构地区：[1]贵州医科大学贵州省药物制剂重点实验室/省部共建药用植物功效与利用国家重点实验室,贵州贵阳550004 [2]贵州医科大学药学院,贵州贵阳550025 [3]贵州医科大学民族药与中药开发应用教育部工程研究中心,贵州贵阳550004

出　　处：《细胞与分子免疫学杂志》2021年第1期73-78,共6页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81603189);贵州省科技计划项目(黔科合基础[2019]1280号);贵州省科学技术厅人才团队项目(黔科合平台人才[2016]5613\5677);中央引导地方科技专项项目(黔科中引地[2018]4006)。

摘　　要：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.

关 键 词：表皮生长因子受体变体Ⅲ(EGFRvⅢ) PD0721 间接ELISA 单链抗体 

分 类 号：R446.61[医药卫生—诊断学] R392.11[医药卫生—临床医学]