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[目的]以合成抗原EGFRvⅢ-BSA作为被检测物,建立检测抗EGFRvⅢ单链抗体的间接ELISA检测方法并优化其反应条件。[方法]利用N-马来酰亚胺甲基环己烷-1-羧酸琥珀酰亚胺酯(SMCC)将EGFRvⅢ与BSA偶联制备抗原。利用方阵滴定法对间接ELISA法的抗原包被浓度,单链抗体的稀释度、包被时间和温度、包被液、抗His-tag多抗稀释度和酶标抗体的稀释度进行优化,并对建立的间接ELISA方法的方法学进行验证。[结果]通过SDS-PAGE电泳和全波长紫外扫面显示EGFRvⅢ-BSA合成成功。用PBS于37℃包被1.25μg/m L的EGFRvⅢ-BSA 2 h,加入19.044μg/L的ChiMR1、1∶8 000的兔抗His-tag多抗和1∶10 000酶标抗体可获得最佳ELISA检测结果。应用优化后的条件检测4.761~152.35μg/L范围内的ChiMR1显示线性良好。线性方程为y=(A-D)/[1+(x/C)^B]+D(A:1.99706;B:-1.09760;C:35.54885;D:0.01558),R2=0.99923;60μg/L和20μg/L的质控品的检测精密度分别为7.142%和4.828%;准确度分别为91.8%和82.90%,准确度和精密度良好。[结论]建立的间接ELISA方法的线性、准确度和精密度良好,为临床疾病的检测奠定良好基础。
[Objective] To establish an indirect ELISA detection method for detecting anti-EGFRv Ⅲ single chain antibody with EGFRvⅢ-BSA as test substance and optimize the reaction conditions. [Method] EGFRv Ⅲ was conjugated with BSA by N-maleimidomethylcyclohexane-1-carboxylic acid succinimidyl ester (SMCC) to prepare antigen. The antigen concentration, the dilution of single-chain antibody, the coating time and temperature, the coating solution, the dilution of anti-His-tag polyclonal antibody and the dilution of enzyme-labeled antibody were optimized by the method of square matrix titration. The methodology of the established indirect ELISA method was validated. [Result] The synthesis of EGFRvⅢ-BSA was successful by SDS-PAGE electrophoresis and full wavelength UV scanning. 1.25μg / ml EGFRvIII-BSA was coated with PBS at 37 ° C for 2 h and added with 19.044μg / L ChiMR1, 1: 8000 rabbit anti-His-tag polyclonal antibody and 1:10000 enzyme labeled antibody ELISA test results. ChiMR1 in the range of 4.761-152.35 μg / L showed good linearity after application of optimized conditions. The linear equations were y = AD / 1 + x / C ^ B] + D (A: 1.99706; B: -1.09760; C: 35.54885; D: 0.01558), R2 = 0.99923; 60 μg / L and 20 μg / L of the quality control of the test were 7.142% and 4.828% precision; accuracy were 91.8% and 82.90%, accuracy and precision. [Conclusion] The established indirect ELISA method had good linearity, accuracy and precision, which laid a good foundation for the detection of clinical diseases.