Q-Bodies from Recombinant Single-Chain Fv Fragment with Better Yield and Expanded Palette of Fluorophores

Abstract

Fluorescence-based immunosensors serve a vital role in biotechnology and diagnostic and therapeutic applications. Our group recently developed a unique fluoroimmunosensor named Quenchbody (Q-body) that operates based on the principle of quenching and the antigen-dependent release of fluorophore, which is incorporated to a recombinant antibody fragment, either the single-chain Fv (scFv) or the Fab fragment of an antibody, using a cell-free transcription-translation system. With the objective of extending the functionality and diversity of the Q-body, here we attempted to make Q-bodies by labeling the recombinant scFv, which was prepared from E. coli using several commercially available dye-maleimides. As a result, we reproducibly obtained larger amounts of antiosteocalcin Q-bodies, with an improved yield and cost-efficiency compared with those obtained from a conventional cell-free system. The fluorescence intensity of each Q-body, including that labeled with newly tested rhodamine red, was significantly increased in the presence of an antigen with a low detection limit, although some differences in response were observed for the dye with different spacer lengths between dye and maleimide. The results indicate the Q-body’s applicability as a powerful multicolored sensor, with a potential to simultaneously monitor multiple targets in a sample.

Development of a fluvoxamine detection system using a Quenchbody, a novel fluorescent biosensor

“…Q‐bodies were primarily created using a cell‐free transcription system . Recently, to improve production yield, Jeong et al developed a method for large‐scale production of Q‐bodies, which combined E. coli expression of recombinant scFv and post‐fluorescence chemical labeling . They prepared a precursor scFv from the soluble fraction of E. coli , and the yield was 400–600 μg/100 mL culture.…”

“…Using pET‐HL as a template, two parts of V H ‐V L type anti‐FLV scFv genes were amplified using two primer pairs (primers 3 and 4; primers 5 and 6). PCR products were digested with Age I and Sac I or Sac I and Bam H I, and the two purified PCR products were inserted into Age I/ Bam H I‐digested pSQ (KTM219) plasmid (an expression vector for anti‐osteocalcin [bone Gla protein] scFv‐type Q‐body) using Ligation high Ver.2, resulting in pSQ (FLV)‐N0.…”

“…To prepare Q‐bodies against FLV, scFv fluorescence labeling and purification was performed using the method of Jeong et al with slight modifications. Briefly, purified scFv (10 μg) and an equal volume of immobilized Tris (2‐carboxyethyl) phosphine (TCEP) disulfide reducing gel slurry were mixed and incubated for 1 hour at 25°C on a rotating wheel.…”

“…To examine the maximum FI of the Q‐bodies, the Q‐body denaturing test was performed as described by Jeong et al using 200 μL of 7 M guanidium hydrochloride (GdnHCl) and 100 mM of dithiothreitol.…”

“…Recently, Ueda et al developed an innovative fluoroimmunosensor named Quenchbody (Q‐body) . Q‐body is a fluorescent antibody or antibody fragment, such as fragment of antigen binding (Fab) or scFv . Fluorophore quenching by tryptophan (Trp) residues in the Q‐body is inhibited when the antigen is bound to Q‐body resulting in de‐quenching in an antigen concentration‐dependent manner.…”