“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.

Enantioselective Monoclonal Antibodies for Detecting Ketamine to Crack Down on Illicit Use

“…The hybridoma clones that we established are also valuable as a source of genetic information for these antibodies, which is necessary for extending next-generation technologies using genetically engineered antibodies. [34][35][36] The genes encoding the variable domains of the antibodies referred above have already been cloned, and relevant single-chain Fv fragments (scFvs) that with KT-binding ability have been prepared in other laboratories (unpublished data). In vitro evolution on these scFvs might create improved antibodies with higher affinity enabling more sensitive assays.…”

Antibody Fragments for On-Site Testing of Cannabinoids Generated <i>via in Vitro</i> Affinity Maturation

“…[15][16][17] Q-body, a family of innovative antibody reagents particularly suitable for on-site testing was recently developed. 18,19) Q-body is antibody fragments (scFvs or Fab fragments) labeled at specific sites with fluorescent dyes, and is usually prepared by in vitro translation. Q-body fluoresces only when bound to cognate antigens, and enables homogenous, noncompetitive, sensitive, and quick detection of small molecules, which is almost impossible to perform with the conventional immunoassays.…”

Synthesis of double-fluorescent labeled prion protein for FRET analysis

“…26) The reaction mixture, containing 500 ng plasmid DNA, 400 pmol B558AF-tRNA, and 400 pmol BFLAF-tRNA, was incubated at 20°C by shaking on a RTS ProteoMaster (Roche Diagnostics, Basel, Switzerland) at 600 rpm for 2 h, and then at 4°C without shaking for 16 h. To purify the labeled protein, the total reaction mixture (50 μL × 6 = 300 μL) was centrifuged at 16,000 × g for 15 min, and 90 μL of the supernatant was diluted in Wash-A buffer (20 mM phosphate, 500 mM NaCl, 30 mM imidazole, 0.1% polyoxyethylene(23)lauryl ether, pH 7.4) to a final volume of 180 μL and was applied to a His SpinTrap column (GE Healthcare, Piscataway, NJ). After incubation at room temperature for 15 min, the column was washed eight times with Wash-A buffer and four times with Wash-B buffer (20 mM phosphate, 500 mM NaCl, 0.1% polyoxyethylene(23)lauryl ether, pH 7.4).…”