Plumer Alicia, Duan Hongyi, Subramaniam Sripriya, Lucas F Lee, Miesfeldt Susan, Ng Ah-Kau, Liaw Lucy
Dept. Applied Medical Sciences, Univ, Southern Maine, P,O, Box 9300 Portland, ME 04104, USA.
BMC Cancer. 2008 Jan 31;8:38. doi: 10.1186/1471-2407-8-38.
Osteopontin (OPN) is associated with human cancers, and circulating blood OPN may have diagnostic or prognostic value in clinical oncology.
To evaluate OPN as a cancer biomarker, we generated and characterized five novel mouse monoclonal antibodies against the human full-length OPN (fl-OPN). Epitopes recognized by four antibodies (2C5, 2F10, 2H9, and 2E11) map to N-terminal OPN (aa1-166); one (1F11) maps to C-terminal OPN (aa167-314). These antibodies recognize recombinant and native OPN by ELISA and immunoblot, cross reacting with human and mouse OPN. Two of these novel antibodies (2F10 and 1F11) were used to develop a quantitative enzyme linked immunosorbent assay (ELISA) for fl-OPN.
In comparison with commercially available ELISAs, our assay had high accuracy in measuring fl-OPN standards, and high sensitivity. Specifically, our ELISA has a linear dose response between 0.078 ng/ml-10 ng/ml, with a sensitivity of 13.9 pg/ml. We utilized this assay to quantify fl-OPN in the plasma of healthy volunteers in comparison with patients with metastatic breast cancer. The average circulating plasma fl-OPN in healthy volunteers was 1.2 ng/ml, compared to 4.76 ng/ml in patients with metastatic breast cancer (p = 0.0042). Although the increase in fl-OPN in cancer patients is consistent with previous studies, the measured quantity varied greatly between all existing fl-OPN ELISAs.
Because OPN is a complex molecule with diversity from alternative splicing, post-translational modification, extracellular proteolytic modification, and participation in protein complexes, we suggest that further understanding of specific isoform recognition of multiple OPN species is essential for future studies of OPN biomarker utility.
骨桥蛋白（OPN）与人类癌症相关，循环血液中的OPN在临床肿瘤学中可能具有诊断或预后价值。
为了评估OPN作为癌症生物标志物的作用，我们制备并鉴定了五种针对人全长OPN（fl-OPN）的新型小鼠单克隆抗体。四种抗体（2C5、2F10、2H9和2E11）识别的表位位于OPN的N端（第1至166位氨基酸）；一种抗体（1F11）识别的表位位于OPN的C端（第167至314位氨基酸）。这些抗体通过ELISA和免疫印迹识别重组和天然OPN，与人源和鼠源OPN发生交叉反应。其中两种新型抗体（2F10和1F11）用于开发fl-OPN的定量酶联免疫吸附测定（ELISA）。
与市售ELISA相比，我们的检测方法在测量fl-OPN标准品时具有较高的准确性和灵敏度。具体而言，我们的ELISA在0.078 ng/ml至10 ng/ml之间具有线性剂量反应，灵敏度为13.9 pg/ml。我们利用该检测方法对健康志愿者和转移性乳腺癌患者血浆中的fl-OPN进行定量。健康志愿者血浆中fl-OPN的平均循环水平为1.2 ng/ml，而转移性乳腺癌患者为4.76 ng/ml（p = 0.0042）。尽管癌症患者中fl-OPN的升高与先前的研究一致，但所有现有fl-OPN ELISA检测到的量差异很大。
由于OPN是一个复杂的分子，存在可变剪接、翻译后修饰、细胞外蛋白水解修饰以及参与蛋白质复合物等多样性，我们建议进一步了解多种OPN异构体的特异性识别对于未来OPN生物标志物效用的研究至关重要。
