title: "Quenchbodies": quench-based antibody probes that show antigen-dependent fluorescence.
abstract: Here, we describe a novel reagentless fluorescent biosensor strategy based on the antigen-dependent removal of a quenching effect on a fluorophore attached to antibody domains. Using a cell-free translation-mediated position-specific protein labeling system, we found that an antibody single chain variable region (scFv) that had been fluorolabeled at the N-terminal region showed a significant antigen-dependent fluorescence enhancement. Investigation of the enhancement mechanism by mutagenesis of the carboxytetramethylrhodamine (TAMRA)-labeled anti-osteocalcin scFv showed that antigen-dependency was dependent on semiconserved tryptophan residues near the V(H)/V(L) interface. This suggested that the binding of the antigen led to the interruption of a quenching effect caused by the proximity of tryptophan residues to the linker-tagged fluorophore. Using TAMRA-scFv, many targets including peptides, proteins, and haptens including morphine-related drugs could be quantified. Similar or higher sensitivities to those observed in competitive ELISA were obtained, even in human plasma. Because of its versatility, this "quenchbody" is expected to have a range of applications, from in vitro diagnostics, to imaging of various targets in situ.
authors: School of Materials Science, Japan Advanced Institute of Science and Technology, Nomi, Ishikawa 923-1292, Japan.
authors: Ryoji
authors: R
authors: Abe
authors: Hiroyuki
authors: H
authors: Ohashi
authors: Issei
authors: I
authors: Iijima
authors: Masaki
authors: M
authors: Ihara
authors: Hiroaki
authors: H
authors: Takagi
authors: Takahiro
authors: T
authors: Hohsaka
authors: Hiroshi
authors: H
authors: Ueda
mesh_terms: D000906:Antibodies / Q000737:chemistry*; D000937:Antigen-Antibody Reactions; D000941:Antigens / Q000737:chemistry*; D015374:Biosensing Techniques; D005453:Fluorescence; D008958:Models, Molecular; D015335:Molecular Probes / Q000737:chemistry*; D015394:Molecular Structure; D049268:Positron-Emission Tomography
chemical_list: D000906:Antibodies; D000941:Antigens; D015335:Molecular Probes