Development of a high-specificity enzyme-linked immunosorbent assay (ELISA) system for the quantification and validation of intact rat osteocalcin.

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 < or =4.9% and