title: Open flower fluoroimmunoassay: a general method to make fluorescent protein-based immunosensor probes.
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 VH and acceptor FP-fused VL 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 VH and VL 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 VH and an acceptor-fused VL linked by a disulfide bond between VH and VL (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.
authors: †Department of Chemistry and Biotechnology, School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
authors: Chan-I
authors: CI
authors: Chung
authors: †Department of Chemistry and Biotechnology, School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
authors: Ryoji
authors: R
authors: Makino
authors: ‡Chemical Resources Laboratory, Tokyo Institute of Technology, 4259-R1-18, Nagatsuta-cho, Midori-ku, Yokoyama, Kanagawa 226-8503, Japan.
authors: Jinhua
authors: J
authors: Dong
authors: ‡Chemical Resources Laboratory, Tokyo Institute of Technology, 4259-R1-18, Nagatsuta-cho, Midori-ku, Yokoyama, Kanagawa 226-8503, Japan.
authors: Hiroshi
authors: H
authors: Ueda
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D015200:Fluoroimmunoassay / Q000379:methods*; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry*; D008958:Models, Molecular; D011487:Protein Conformation; D012709:Serum Albumin / Q000032:analysis; D061905:Single-Domain Antibodies / Q000737:chemistry / Q000276:immunology
chemical_list: D004220:Disulfides; D008164:Luminescent Proteins; D012709:Serum Albumin; D061905:Single-Domain Antibodies