Department of Chemistry, University of Colorado Denver, Denver, CO 80217-3364, USA.
Anal Bioanal Chem. 2011 Sep;401(4):1309-18. doi: 10.1007/s00216-011-5174-1. Epub 2011 Jul 2.
Native C-reactive protein (CRP) is composed of five identical subunits arranged in a pentameric structure (pCRP). Binding of pCRP to damaged cell membranes produces a second isoform, modified CRP, which has similar antigenicity to isolated monomeric subunits of CRP (mCRP). Emerging evidence indicates that modified CRP plays a role in inflammation and atherosclerosis, however, there are very few techniques that can distinguish the different isoforms of CRP. Here we show that an RNA aptamer binds specifically to mCRP and not to pCRP. Using this aptamer, we describe a simple, fast, and sensitive assay to detect nanomolar concentrations of mCRP using fluorescence anisotropy. In addition, we show that this aptamer can be used to detect mCRP in polyacrylamide gels and bound to a surface using total internal reflection fluorescence microscopy. The biological activity of the mCRP we prepared by heating pCRP with 0.1% sodium dodecyl sulfate was confirmed by observing binding to the complement protein, C1q. This probe provides an important tool for CRP research and has the potential to improve clinical diagnostics that predict risk for cardiovascular disease.
天然 C 反应蛋白（CRP）由五个相同的亚基组成，呈五聚体结构（pCRP）。pCRP 与受损细胞膜结合产生第二种同工型，即修饰型 CRP，其与 CRP 分离的单体亚基（mCRP）具有相似的抗原性。新出现的证据表明，修饰型 CRP 在炎症和动脉粥样硬化中起作用，但是，能够区分 CRP 不同同工型的技术却很少。在这里，我们展示了一种 RNA 适体可以特异性地结合 mCRP，而不与 pCRP 结合。使用这种适体，我们描述了一种使用荧光各向异性检测纳摩尔浓度 mCRP 的简单、快速和灵敏的测定方法。此外，我们还表明，该适体可以用于检测聚丙烯酰胺凝胶中的 mCRP，并通过全内反射荧光显微镜检测到与表面的结合。我们通过用 0.1％十二烷基硫酸钠加热 pCRP 制备的 mCRP 的生物学活性通过观察与补体蛋白 C1q 的结合得到证实。该探针为 CRP 研究提供了重要工具，并有可能改善预测心血管疾病风险的临床诊断。
