title: Development of a fluorescent protein-antibody Förster resonance energy transfer probe for the detection and imaging of osteocalcin.
abstract: Fluorescence-based biosensor probes, especially those based on Förster resonance energy transfer (FRET) between fluorescent protein (FP) variants, are widely used to monitor various biological phenomena, often detecting ligand-induced association of the receptor domains. While antibodies are fertile sources of specific receptors for various biomolecules, their potential has not been fully exploited. In this study, we used a fluorescent probe comprising FP-fused antibody variable region fragments to detect a bone metabolism biomarker, osteocalcin (BGP), by using fluorescence spectrometry/microscopy. Because the association between the two proteins increases in the presence of antigen BGP or its C-terminal peptide, the increased antigen in a sample can be monitored as a FRET efficiency increase, based on the open sandwich fluoroimmunoassay principle. The results clearly indicated that the FP-antibody FRET probe could be used as a diagnostic reagent to measure levels of BGP in the clinically relevant concentration range and to image BGP produced from live osteoblast cells.
authors.affiliation: Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, 4259-R1-18 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8503, Japan; Department of Chemistry and Biotechnology, School of Engineering, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
authors.forename: Chan-I
authors.initials: CI
authors.lastname: Chung
authors.affiliation: Department of Chemistry and Biotechnology, School of Engineering, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
authors.forename: Ryoji
authors.initials: R
authors.lastname: Makino
authors.affiliation: Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, 4259-R1-18 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8503, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Ohmuro-Matsuyama
authors.affiliation: Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, 4259-R1-18 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8503, Japan; Department of Chemistry and Biotechnology, School of Engineering, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan. Electronic address: ueda@res.titech.ac.jp.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Ueda
mesh_terms: D000906:Antibodies / Q000032:analysis; D015374:Biosensing Techniques / Q000379:methods; D002478:Cells, Cultured; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000737:chemistry; D006801:Humans; D007118:Immunoassay / Q000379:methods; D007135:Immunoglobulin Variable Region / Q000737:chemistry; D008024:Ligands; D008856:Microscopy, Fluorescence / Q000379:methods; D057054:Molecular Imaging / Q000379:methods*; D015675:Osteocalcin / Q000032:analysis* / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: D000906:Antibodies; D005456:Fluorescent Dyes; D007135:Immunoglobulin Variable Region; D008024:Ligands; D015675:Osteocalcin
keywords: Bioimaging; Fluorescent protein; Förster resonance energy transfer; Homogenous immunoassay; Noncompetitive