Gudmann Natasja Stæhr, Wang Jianxia, Hoielt Sabine, Chen Pingping, Siebuhr Anne Sofie, He Yi, Christiansen Thorbjørn G, Karsdal Morten Asser, Bay-Jensen Anne Christine
Nordic Bioscience A/S, Herlev Hovedgade 207, DK-2730 Herlev, Denmark.
Orthopedic Department Z, Gentofte University Hospital, Niels Andersensvej 65, DK-2900 Hellerup, Denmark.
Int J Mol Sci. 2014 Oct 17;15(10):18789-803. doi: 10.3390/ijms151018789.
The aim of this study was to enable measurement of cartilage formation by a novel biomarker of type II collagen formation. The competitive enzyme-linked immunosorbent assay (ELISA) Pro-C2 was developed and characterized for assessment of the beta splice variant of type II procollagen (PIIBNP). This is expected to originate primarily from remodeling of hyaline cartilage. A mouse monoclonal antibody (Mab) was raised in mouse, targeting specifically PIIBNP (QDVRQPG) and used in development of the assay. The specificity, sensitivity, 4-parameter fit and stability of the assay were tested. Levels of PIIBNP were quantified in human serum (0.6-2.2 nM), human amniotic fluid (163-188 nM) and sera from different animal species, e.g., fetal bovine serum (851-901 nM) with general good linearity (100% (SD 7.6) recovery) and good intra- and inter-assay variation (CV% < 10). Dose (0.1 to 100 ng/mL) and time (7, 14 and 21 days) dependent release of PIIBNP were evaluated in the conditioned medium from bovine cartilage explants (BEX) and human cartilage explants (HEX) upon stimulation with insulin-like growth factor (IGF-1), transforming growth factor (TGF)-β1 and fibroblastic growth factor-2 (FGF-2). TGF-β1 and IGF-1 in concentrations of 10-100 ng/mL significantly (p < 0.05) induced release of PIIBNP in BEX compared to conditions without treatment (WO). In HEX, IGF-1 100 ng/mL was able to induce a significant increase of PIIBNP after one week compared to WO. FGF-2 did not induce a PIIBNP release in our models. To our knowledge this is the first assay, which is able to specifically evaluate PIIBNP excretion. The Pro-C2 assay seems to provide a promising and novel marker of type II collagen formation.
本研究的目的是通过一种新型的II型胶原蛋白形成生物标志物来实现软骨形成的测量。开发并表征了用于评估II型前胶原蛋白（PIIBNP）β剪接变体的竞争性酶联免疫吸附测定（ELISA）Pro-C2。预计其主要源自透明软骨的重塑。在小鼠中制备了一种小鼠单克隆抗体（Mab），该抗体特异性靶向PIIBNP（QDVRQPG）并用于该测定的开发。测试了该测定的特异性、灵敏度、四参数拟合和稳定性。对人血清（0.6 - 2.2 nM）、人羊水（163 - 188 nM）以及来自不同动物物种的血清（例如胎牛血清（851 - 901 nM））中的PIIBNP水平进行了定量，总体线性良好（回收率100%（标准差7.6）），且批内和批间变异良好（CV% < 10）。在用胰岛素样生长因子（IGF - 1）、转化生长因子（TGF） - β1和成纤维细胞生长因子 - 2（FGF - 2）刺激后，评估了牛软骨外植体（BEX）和人软骨外植体（HEX）的条件培养基中PIIBNP的剂量（0.1至100 ng/mL）和时间（7、14和21天）依赖性释放。与未处理（WO）条件相比，浓度为10 - 100 ng/mL的TGF - β1和IGF - 1显著（p < 0.05）诱导BEX中PIIBNP的释放。在HEX中，与WO相比，100 ng/mL的IGF - 1在一周后能够诱导PIIBNP显著增加。FGF - 2在我们的模型中未诱导PIIBNP释放。据我们所知，这是第一个能够特异性评估PIIBNP排泄的测定方法。Pro - C2测定似乎提供了一种有前景的新型II型胶原蛋白形成标志物。
