Ultra Q-bodies: quench-based antibody probes that utilize dye-dye interactions with enhanced antigen-dependent

Recently, we described a novel reagentless fluorescent biosensor strategy named Quenchbody, whichfunctions via the antigen-dependent removal of the quenching effect on a fluorophore that is attached to asingle-chain antibody variable region. To explore the practical utility of Quenchbodies, we preparedantibody Fab fragments that were fluorolabeled at either one or two of the N-terminal regions, using acell-free translation-mediated position-specific protein labeling system. Unexpectedly, the Fab fragmentlabeled at the heavy chain N-terminal region demonstrated a deeper quenching and antigen-dependentrelease compared to that observed using scFv. Moreover, when the Fab was fluorolabeled at the twoN-termini with either the same dye or with two different dyes, an improved response due to enhancedquenching via dye-dye interactions was observed. On the basis of this approach, several targets, includingpeptides,proteins,andhaptens,aswellasnarcotics,werequantifiedwithahigherresponseupto50-fold.Inaddition, differentiation of osteosarcoma to osteoblasts was successfully imaged using a similarlyfluorolabeled recombinant Fab protein prepared from E. coli. Due to its versatility, this‘‘Ultra-Quenchbody’’ is expected to exhibit a range of applications from in vitrodiagnostics to the liveimaging of various targets in situ.