A competitive fluorescence immunoassay based on intermolecular quenching using an N-terminally fluorescent-labeled...

The development of antibody-based fluorescent sensors relying on tryptophan-mediated quenching, such as Quenchbody (Q-body), often exhibits limited fluorescence responses because dye quenching depends on the location of tryptophan residues within the antibody. Here, we developed a competitive fluorescence immunoassay, termed an intermolecular Quenchbody (iQ-body), that utilizes intermolecular Förster resonance energy transfer (FRET) between an N-terminally fluorescent-labeled IgG antibody and an antigen-quencher conjugate. Anti-thyroxine IgG antibody (clone 6901 SPTN-5) showed minimal fluorescence changes upon antigen binding, despite N-terminal labeling with TAMRA. In contrast, the addition of an antigen-quencher conjugate (QSY9-X-T3) effectively quenched the TAMRA fluorescence via intermolecular FRET. Subsequent competitive displacement by thyroxine (T4) resulted in concentration-dependent fluorescence recovery. The iQ-body strategy provides a simple approach for constructing competitive fluorescence immunoassays for small-molecule targets using publicly available IgG antibodies.