Department of Public Health, Health Management and Policy, Nara Medical University School of Medicine, 840 Shijo-cho, Kashihara, Nara, 634-8521, Japan.
Immunol Invest. 2010;39(1):54-73. doi: 10.3109/08820130903428283.
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 </=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.
骨钙素（OC）表现出组织特异性表达和与羟磷灰石的结合活性。因此，分泌型 OC 的测量是评估再生骨中成骨细胞分化的非常有用的指标。在本研究中，我们建立了一种用于定量检测完整大鼠 OC 的高特异性夹心酶联免疫吸附测定（ELISA）系统，该系统可用于非破坏性和连续验证组织工程骨样本。夹心 ELISA 系统的检测范围为 0.1-100ng OC/mL 细胞培养液或大鼠血清。与来自其他物种的 OC（包括人、牛和鼠 OC）以及其他含相应内源性 OC 的哺乳动物血清没有交叉反应。内和间测定的变异系数分别为<或=4.9%和</=5.9%。回收试验仅显示 89.4%和 103.7%之间的差异。使用新开发的直接夹心 ELISA 系统，我们发现地塞米松诱导大鼠骨髓间充质干细胞成骨分化过程中分泌的 OC 水平明显高于非成骨或成脂分化的细胞。该 ELISA 系统可适用于大鼠实验模型中有无支架的培养细胞定量评估骨形成。
