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

Abstract

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