Osteocalcin is a bone-specific protein which contains three glutamic acid residues (Glu) that undergo post-translational gamma-carboxylation. Uncarboxylated osteocalcin (ucOC) may participate in the regulation of glucose metabolism, thus measurement of ucOC could be useful in evaluating interactions between bone and glucose metabolism. We developed recombinant antibodies and immunoassay to specifically detect ucOC in human blood samples. ucOC-specific recombinant antibodies were selected from an antibody library by phage display. Four candidates were characterized, and one (Fab-AP13) was used to set up an immunoassay with a pre-existing MAb. Plasma ucOC levels were measured in subjects with normal fasting blood glucose (≤ 6 mmol/l, N = 46) or with hyperglycemia (≥ 7 mmol/l, N = 29). Further, we analyzed ucOC in age- and gender-matched patients with diagnosed type 2 diabetes (T2D, N = 49). Antibodies recognized ucOC without cross-reaction to carboxylated osteocalcin. Antibodies had unique binding sites at the carboxylation region, with Glu17 included in all epitopes. Immunoassay was set up and characterized. Immunoassay detected ucOC in serum and plasma, with on average 1.6-fold higher levels in plasma. ucOC concentrations were significantly lower in subjects with hyperglycemia (median 0.58 ng/ml, p = 0.008) or with T2D diagnosis (0.68 ng/ml, p = 0.015) than in subjects with normal blood glucose (1.01 ng/ml). ucOC negatively correlated with fasting plasma glucose in subjects without T2D (r = − 0.24, p = 0.035) but not in T2D patients (p = 0.41). Our immunoassay, based on the novel recombinant antibody, allows for specific and sensitive detection of ucOC in human circulation. Correlation between ucOC and plasma glucose suggests interactions between osteocalcin and glucose metabolism in humans.

骨钙素是一种骨特异性蛋白，其中包含三个谷氨酸残基（Glu），这些残基会进行翻译后的γ-羧化反应。未羧化骨钙素（ucOC）可能参与葡萄糖代谢的调节，因此ucOC的测量可能有助于评估骨骼与葡萄糖代谢之间的相互作用。我们开发了重组抗体和免疫测定法来特异性检测人血样品中的ucOC。通过噬菌体展示从抗体文库中选择ucOC特异性重组抗体。表征了四个候选物，其中一个（Fab-AP13）用于建立具有预先存在的MAb的免疫测定。在正常禁食血糖（≤6 mmol / l，N  = 46）或高血糖（≥7 mmol / l，N）的受试者中测量血浆ucOC水平 = 29）。此外，我们分析了年龄和性别匹配的诊断为2型糖尿病（T2D，N  = 49）的患者的ucOC 。抗体识别出ucOC不会与羧化骨钙素发生交叉反应。抗体在羧基化区域具有独特的结合位点，所有表位均包含Glu17。建立了免疫测定法并进行了表征。免疫测定法检测到的血清和血浆中的ucOC，血浆中的ucOC水平平均高1.6倍。高血糖（中位数0.58 ng / ml，p  = 0.008）或经T2D诊断的 受试者（0.68 ng / ml，p = 0.015）的ucOC浓度显着低于正常血糖（1.01 ng / ml）的受试者。在没有T2D的受试者中，ucOC与空腹血糖呈负相关（r  = − 0.24，p  = 0.035），但在T2D患者中则没有（p  = 0.41）。我们基于新型重组抗体的免疫分析方法可对人体内的ucOC进行特异性和灵敏的检测。ucOC与血浆葡萄糖之间的相关性暗示了人骨钙素与葡萄糖代谢之间的相互作用。