Quenchbodies are a novel antibody derivative with a fluorescent tag close to the complementarity determining regions [1]. This fluorescent dye exists in a quenched state when the antibody is unbound (apo-state) due to photoinduced electron transfer with intrinsic tryptophan residues. When bound to cognate antigen quenching is disrupted, resulting in an increase in fluorescence. This signal can be used to measure analyte concentration in solution. Quenchbodies offer several advantages over traditional antibody based fluorometric assays by combining capture and detection events, in particular the removal of necessary wash steps.
Quenchbodies developed in our lab have been shown to have robust response to their cognate antigen in bulk. Bulk assays are the standard by which analyte concentration measurements are done but they require relatively large amount of sample. We investigated the possibility of using total internal reflection microscopy and single molecule techniques to determine analyte concentration in solution using surface tethered quenchbodies