One-step non-competitive fluorescence polarization immunoassay based on a Fab fragment for C-reactive protein quantification

A non-competitive fluorescence polarization immunoassay (FPIA) using a Fab fragment was developed for large molecule quantification. Most conventional FPIAs are homogeneous and competitive immunoassays. With FPIAs, it has been difficult to obtain sufficient detection sensitivity when targeting molecules like proteins. To overcome this fundamental drawback, we report FPIA fluorescence-labeled fragment. C-reactive protein (CRP) used as model substance validation of the Fab-based FPIA. Quantitative analysis CRP in phosphate buffered saline (PBS) successfully achieved limit (LOD) PBS 207 ng/mL. Moreover, far-red emitting fluorescent dye (HiLyte Fluor™647) labeling fragment, measured human serum without pretreatment sample 10 min around cut-off value (10 μg/mL). The LOD 1.58 μg/mL. In our proposed method, reaction completed simple one-step mixing with only one type reagent, no washing operation required. Therefore, rapid quantification greatly simplified procedure.