Development of a High-specificity Enzyme-linked Immunosorbent Assay (ELISA) System for the Quantification and Validation of Intact Rat Osteocalcin

Abstract

Osteocalcin (OC) exhibits hard tissue-specific expression and binding activity to hydroxyapatite. Therefore, measurement of secreted OC is a very useful index for evaluating osteoblastic differentiation in regenerative bone. In the present study, we established a high-specificity sandwich enzyme-linked immunosorbent assay (ELISA) system for the quantification of intact rat OC, which could be useful for validating tissue-engineered bone samples nondestructively and continuously. The range of detection with the sandwich ELISA system was 0.1–100 ng OC/mL of cell culture media or rat sera. No cross-reactivities were detected with OCs from other species, including human, bovine and mouse OCs, and other mammalian sera, which would contain the corresponding endogenous OCs. The intra- and inter-assay coefficients of variation were ≤4.9% and ≤5.9%, respectively. Recovery tests only showed variation between 89.4% and 103.7%. Using the newly developed direct sandwich ELISA system, we found that the secreted OC levels from rat bone marrow-derived mesenchymal stem cells during osteogenic differentiation with dexamethasone were significantly higher than those from cells undergoing non-osteogenic or adipogenic differentiation. It was established that this ELISA system would be suitable for quantitative assessment of bone formation by cultured cells with or without scaffolds in rat experimental models.

Keywords:

• Osteocalcin
• Rat mesenchymal stem cell (MSC)
• Sandwich enzyme-linked immunosorbent assay (ELISA)
• Monoclonal antibody
• Quantification

ACKNOWLEDGMENTS

We thank Nara Prefecture for a Grant-in-Aid for Academic-Industrial Research Relationships and Ms. M. Yoshimura for her technical assistance.

Declaration of Interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.