An Indirect Immunoassay for Detecting Antigen Based on Fluorescence Resonance Energy Transfer

An indirect immunoassay for detecting antigen was developed. It was based on fluorescence resonance energy transfer (FRET) and quenching of gold nanoparticles. Bovine serum albumin (BSA) was chosen as model antigen. Fluorescein isothiocyanate (FITC) was attached to anti-BSA antibody (anti-BSA–FITC) as FRET donor, while BSA was conjugated to gold nanoparticles (GNPs–BSA) as FRET acceptor. The formation of anti-BSA–BSA immunocomplex resulted in the FRET between anti-BSA–FITC and GNPs–BSA. Thus, the fluorescence of FRET donor was quenched, and the decreasing fluorescence intensity responded linearly to the concentration of acceptor within the linear range. The concentration of BSA we obtained according to the stoichiometric ratio between BSA and GNPs. Following this approach, we were able to specifically detect BSA. The detection limit for BSA was 0.5 nM and the linear range of the assay was 2.9 - 43.5 nM. It had been successfully applied to specific detection of BSA in serum samples.