title: Insight into the Working Mechanism of Quenchbody: Transition of the Dye around Antibody Variable Region That Fluoresces upon Antigen Binding.
abstract: Recently, we reported a novel immunoassay reagent Quenchbody (Q-body): a single chain antibody variable region (scFv) fragment labeled with fluorescent dye, whose fluorescence intensity increases when it binds to the antigen. Here we analyze its working mechanism by immuno- and fluorescence polarization (FP) assays. In an enzyme-linked immunosorbent assay, we found that in the presence of antigen osteocalcin peptide (BGP-C7), more TAMRA-labeled Q-bodies bound to anti-TAMRA antibody than in its absence. Moreover, we found that anti-BGP Q-body with the shortest linker that exhibits the largest antigen-dependency in fluorescence showed the highest binding signal. Similar results were obtained with anti-bisphenol A (BPA) Q-bodies, with inversed correlation with their linker lengths. In the FP assay, when the ATTO 520 labeled Q-body was added with antigen, the Brownian motion of the dye became more active, which resulted in reduced fluorescence anisotropy r. In other words, in the presence of antigen, 1/r showing that the dye mobility is larger than in the absence of its antigen. In addition, anti-BGP Q-body with the largest antigen-dependency in fluorescence showed the highest mobility. Overall, these results clearly suggest that the antigen-dependent fluorescence quenching and recovery of Q-body is caused by the movement of the dye within and around scFv, which moves out of scFv upon binding with its antigen.
authors.affiliation: Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo , 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.|Ushio Incorporated , 6409 Motoishikawa-cho, Aoba-ku, Yokohama 225-0004, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Ohashi
authors.affiliation: Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo , 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Matsumoto
authors.affiliation: Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo , 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.|Laboratory for Chemistry and Life Science, Tokyo Institute of Technology , 4259-R1-18, Nagatsuta-cho, Midori-ku, Yokoyama, Kanagawa 226-8503, Japan.
authors.forename: Hee-Jin
authors.initials: HJ
authors.lastname: Jeong
authors.affiliation: Laboratory for Chemistry and Life Science, Tokyo Institute of Technology , 4259-R1-18, Nagatsuta-cho, Midori-ku, Yokoyama, Kanagawa 226-8503, Japan.
authors.forename: Jinhua
authors.initials: J
authors.lastname: Dong
authors.affiliation: Ushio Incorporated , 6409 Motoishikawa-cho, Aoba-ku, Yokohama 225-0004, Japan.
authors.forename: Ryoji
authors.initials: R
authors.lastname: Abe
authors.affiliation: Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo , 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.|Laboratory for Chemistry and Life Science, Tokyo Institute of Technology , 4259-R1-18, Nagatsuta-cho, Midori-ku, Yokoyama, Kanagawa 226-8503, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Ueda
mesh_terms: D001559:Benzhydryl Compounds / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005454:Fluorescence Polarization / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D008694:Methamphetamine / Q000276:immunology; D015675:Osteocalcin / Q000737:chemistry / Q000378:metabolism; D010636:Phenols / Q000737:chemistry; D012235:Rhodamines / Q000737:chemistry / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000737:chemistry; D014255:Trichothecenes / Q000276:immunology; D000098826:Bisphenol A Compounds
chemical_list: D001559:Benzhydryl Compounds; D005456:Fluorescent Dyes; D008694:Methamphetamine; D015675:Osteocalcin; D010636:Phenols; D012235:Rhodamines; D057127:Single-Chain Antibodies; D014255:Trichothecenes; C114557:5-carboxytetramethylrhodamine succinimidyl ester; C007262:deoxynivalenol; C006780:bisphenol A; D000098826:Bisphenol A Compounds