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Crystal Structures of Antigen-Binding Fragment of Anti-Osteocalcin Antibody KTM219
Shuma Yazaki1, Misaki Komatsu1, Jinhua Dong2
1Department of Applied Biology, Faculty of Textile Science and Technology, Shinshu University, Ueda 386-8567, Nagano, Japan.
Abstract:
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.
Insights
This study reveals the crystal structure of an anti-osteocalcin antibody fragment (Fab) and its antigen. These findings support the mechanism of Quenchbody (Q-body) fluorescent immunosensors for detecting bone disease biomarkers.
Area of Science:
- Biochemistry
- Structural Biology
- Immunosensors
Background:
- Osteocalcin is a key biomarker for bone formation and related diseases.
- KTM219 is an antibody targeting the C-terminal peptide of osteocalcin.
- The single-chain variable fragment (scFv) and antigen-binding fragment (Fab) of KTM219 are utilized in Quenchbody (Q-body) immunoassays.
Purpose of the Study:
- To elucidate the recognition mechanism of the KTM219 antibody.
- To provide the structural basis for the Quenchbody (Q-body) fluorescent immunosensor technology.
- To understand the structural interactions between KTM219 Fab and its osteocalcin antigen.
Main Methods:
- X-ray crystallography was employed to determine the structures of KTM219 Fab and its complex with osteocalcin C-terminal peptide (BGP-C7).
- Crystallization of KTM219 Fab in the presence of a fluorescent dye (TAMRA) was performed.
- Molecular docking simulations were used to predict potential binding sites of TAMRA.
Main Results:
- The crystal structures of KTM219 Fab and its complex with BGP-C7 were successfully solved.
- No tightly bound TAMRA was observed in the electron density map of the KTM219 Fab crystal.
- Docking simulations suggested potential binding sites for TAMRA within the antigen-binding pocket.
Conclusions:
- The determined crystal structures provide insights into the KTM219 antibody's recognition mechanism.
- The findings support the proposed mechanism of Q-body fluorescent immunosensors.
- These structural insights are valuable for the future development and optimization of Q-body technology for sensitive biomarker detection.
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