Open Flower Fluoroimmunoassay: A General Method To Make Fluorescent Protein-Based Immunosensor Probes

Fluorescence-based probes, especially those that utilize Förster resonance energy transfer (FRET) between fluorescent protein (FP) variants, are widely used to monitor various biological phenomena, most often detecting its ligand-induced conformational change through the receptor domain. While antibody provides a fertile resource of a specific receptor for various biomolecules, its potential has not been fully exploited. An exception is a pair of donor FP-fused V_H and acceptor FP-fused V_L fragments, which has been proven useful when their association increases in the presence of antigen (open sandwich fluoroimmunoassay, OS-FIA). However, probes for larger proteins such as serum albumin (SA) were difficult to produce, since the interaction between V_H and V_L of these antibodies is barely affected by the bound antigen. Here, we propose a novel strategy, called open flower fluoroimmunoassay (OF-FIA), using a probe composed of a donor-fused V_H and an acceptor-fused V_L linked by a disulfide bond between V_H and V_L (CyPet/YPet-dsFv). The probe gave high FRET efficiency due to the dimerization propensity of the FP pair, while the efficiency got lower as SA concentration increased, probably due to dimer disruption. The constructed probe could detect clinically relevant range of SA, showing its potential as a diagnostic reagent.