Crystal Structures of Antigen-Binding Fragment (Fab) of Anti-Osteocalcin Antibody KTM219: Structural Basis for Quenchbody (Q-body)

Quenchbody (Q-body), a new type of fluorescent immunosensor, is an antibody fragment labeled with a fluorescent dye. When Q-body binds to its antigen, the fluorescence intensity increases. Highly sensitive detection of antigens by changes in fluorescence intensity is performed in a single step by mixing sample and reagent. In this study, to reveal the structural basis for Q-body, we solved the crystal structures of antigen-binding fragment (Fab) of an anti-osteocalcin antibody KTM219, which is applicable to Q-body, and its complex with the antigen osteocalcin C-terminal peptide (BGP-C7). Also, we solved the structure of a KTM219 Fab crystal grown in the presence of a fluorescent dye 5(6)-carboxytetramethylrhodamine (TAMRA), however, a tightly bound TAMRA was not found in the electron density map. We predicted the binding site of TAMRA by docking simulations. These results support the structural basis for the Q-body mechanism as follows. In the absence of the antigen, the fluorophore located near tryptophan residues is quenched in the hydrophobic pocket of the antigen binding site between VH and VL. In association with the competitive binding of the antigen, the fluorophore is released from the antigen binding pocket and emits fluorescence. The crystal structures of KTM219 Fab and the structural basis of Q-body would be useful for further development and improvement of Q-body fluorescent immunosensors.