Open Flower Fluoroimmunoassay: A General Method To
Make Fluorescent Protein-Based Immunosensor Probes
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Abstract
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<sub>H</sub> and acceptor FP-fused V<sub>L</sub> 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<sub>H</sub> and V<sub>L</sub> 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<sub>H</sub> and an acceptor-fused
V<sub>L</sub> linked by a disulfide bond between V<sub>H</sub> and
V<sub>L</sub> (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