Department of Genetics and Pathology, Uppsala University, Uppsala, Sweden.
Mol Cell Proteomics. 2010 Feb;9(2):327-35. doi: 10.1074/mcp.M900248-MCP200. Epub 2009 Nov 27.
Detection of proteins released in the bloodstream from tissues damaged by disease can promote early detection of pathological conditions, differential diagnostics, and follow-up of therapy. Despite these prospects and a plethora of candidate biomarkers, efforts in recent years to establish new protein diagnostic assays have met with limited success. One important limiting factor has been the challenge of detecting proteins present at trace levels in complex bodily fluids. To achieve robust, sensitive, and specific detection, we have developed a microparticle-based solid-phase proximity ligation assay, dependent on simultaneous recognition of target proteins by three antibody molecules for added specificity. After capture on a microparticle, solid-phase pairs of proximity probes are added followed by washes, enabling detection and identification of rare protein molecules in blood while consuming small amounts of sample. We demonstrate that single polyclonal antibody preparations raised against target proteins of interest can be readily used to establish assays where detection depends on target recognition by three individual antibody molecules, recognizing separate epitopes. The assay was compared with state-of-the-art sandwich ELISAs for detection of vascular endothelial growth factor, interleukin-8 and interleukin-6, and it was found to be superior both with regard to dynamic range and minimal numbers of molecules detected. Furthermore, the assays exhibited excellent performance in undiluted plasma and serum as well as in whole blood, producing comparable results for nine different antigens. We thus show that solid-phase proximity ligation assay is suitable for validation of a variety of protein biomarkers over broad dynamic ranges in clinical samples.
从疾病损伤的组织中释放到血液中的蛋白质的检测可以促进对病理状况的早期检测、鉴别诊断和治疗监测。尽管前景广阔，且有大量候选生物标志物，但近年来建立新的蛋白质诊断检测方法的努力收效甚微。一个重要的限制因素是检测复杂体液中痕量蛋白质的挑战。为了实现稳健、灵敏和特异性的检测，我们开发了一种基于微粒的固相邻近连接检测法，该方法依赖于三种抗体分子对目标蛋白的同时识别，以增加特异性。在微粒上捕获后，添加固相邻近探针对，然后进行洗涤，从而能够在消耗少量样本的情况下，检测和鉴定血液中的稀有蛋白质分子。我们证明，针对感兴趣的目标蛋白的单克隆抗体制剂可以很容易地用于建立依赖于三个单独的抗体分子识别目标的检测方法，识别不同的表位。该检测法与最先进的夹心 ELISA 相比，用于检测血管内皮生长因子、白细胞介素-8 和白细胞介素-6，发现它在动态范围和检测到的最小分子数量方面都具有优势。此外，该检测法在未稀释的血浆和血清以及全血中均表现出优异的性能，对 9 种不同的抗原产生了可比的结果。因此，我们表明固相邻近连接检测法适用于在临床样本中对广泛动态范围内的各种蛋白质生物标志物进行验证。
