Department of Materials Science and Engineering, Yonsei University, Seoul, Korea.
Institute of Pharmaceutical and Medical Chemistry, University of Münster, Münster, Germany.
Analyst. 2017 May 21;142(10):1720-1728. doi: 10.1039/c6an02386j. Epub 2017 Apr 12.
Escherichia coli cells that autodisplay Z-domains have been used to improve the sensitivity and limit of detection (LOD) of immunoassays by controlling antibody orientation. In this study, a rapid, wash-free immunoassay was developed using E. coli cells with autodisplayed Z-domains and flow cytometry. The fluorescence signal from a single E. coli cell could be identified and background noise from non-bound detection antibodies was minimal. Flow cytometric measurement was demonstrated to be more effective than microscopy or fluorescence photometry. The concentrations of the capture and detection antibodies were optimized by testing various concentrations; the incubation time was optimized in the same way. The wash-free immunoassay was used to quantify the C-reactive protein (CRP) and the results were compared with those of an established assay method. The results of the wash-free immunoassay were significantly correlated with the results of the washing method, but showed an improved LOD and dynamic range. Thus, the wash-free immunoassay based on E. coli cells with autodisplayed Z-domains and flow cytometry developed here was confirmed to be feasible for use in medical diagnosis.
自展示 Z 结构域的大肠杆菌细胞已被用于通过控制抗体定向来提高免疫分析的灵敏度和检测限 (LOD)。在这项研究中，使用自展示 Z 结构域的大肠杆菌细胞和流式细胞术开发了一种快速、无需洗涤的免疫分析方法。可以识别单个大肠杆菌细胞的荧光信号，并且非结合检测抗体的背景噪声最小。流式细胞术测量被证明比显微镜或荧光光度法更有效。通过测试各种浓度来优化捕获和检测抗体的浓度；同样优化孵育时间。使用无洗涤免疫分析来定量 C 反应蛋白 (CRP)，并将结果与已建立的测定方法进行比较。无洗涤免疫分析的结果与洗涤方法的结果显著相关，但显示出改善的 LOD 和动态范围。因此，基于自展示 Z 结构域的大肠杆菌细胞和流式细胞术开发的无洗涤免疫分析被证实可用于医学诊断。
