Development of a fluorescent protein-antibody Förster resonance energy transfer probe for the detection and imaging of osteocalcin

Fluorescence-based biosensor probes, especially those based on Förster resonance energy transfer (FRET) between fluorescent protein (FP) variants, are widely used to monitor various biological phenomena, often detecting ligand-induced association of the receptor domains. While antibodies are fertile sources of specific receptors for various biomolecules, their potential has not been fully exploited. In this study, we used a fluorescent probe comprising FP-fused antibody variable region fragments to detect a bone metabolism biomarker, osteocalcin (BGP), by using fluorescence spectrometry/microscopy. Because the association between the two proteins increases in the presence of antigen BGP or its C-terminal peptide, the increased antigen in a sample can be monitored as a FRET efficiency increase, based on the open sandwich fluoroimmunoassay principle. The results clearly indicated that the FP-antibody FRET probe could be used as a diagnostic reagent to measure levels of BGP in the clinically relevant concentration range and to image BGP produced from live osteoblast cells.