Crystal Structures of Antigen-Binding Fragment of Anti-Osteocalcin Antibody KTM219

Osteocalcin is a useful biomarker for bone formation and bone-related diseases. KTM219 is an anti-osteocalcin C-terminal peptide antibody. The single-chain variable region (scFv) and antigen-binding fragment (Fab) of KTM219 are applicable to the Quenchbody (Q-body) immunoassay. Q-body is a new type of fluorescent immunosensor, which is scFv or Fab labeled with a fluorescent dye. When Q-body binds to its antigen, the fluorescence intensity increases. The highly sensitive detection of antigens by changes in fluorescence intensity is performed in a single step by mixing the sample and reagent. In this study, to reveal the recognition mechanism of the KTM219 antibody and to discuss the structural basis for Q-body, we solved the crystal structures of Fab of the anti-osteocalcin antibody KTM219 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, carboxytetramethylrhodamine (TAMRA); however, tightly bound TAMRA was not found in the electron density map. We predicted the binding sites of TAMRA in the antigen-binding pocket by docking simulations. These results support the proposed Q-body mechanism. The crystal structures of KTM219 Fab would be useful for further development and improvement of Q-body fluorescent immunosensors.