Development of a wash-free immunoassay using: Escherichia coli cells with autodisplayed Z-domains

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.