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“Quenchbodies”: Quench-Based Antibody Probes That Show Antigen-Dependent Fluorescence

2011; American Chemical Society; Volume: 133; Issue: 43 Linguagem: Inglês

10.1021/ja205925j

1943-2984

Ryoji Abe, Hiroyuki Ohashi, Issei Iijima, Masaki Ihara, Hiroaki Takagi, Takahiro Hohsaka, Hiroshi Ueda,

Biotin and Related Studies

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 VH/VL 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.