Sensitive Plasma Protein Analysis by Microparticle-based Proximity Ligation Assays
Sensitive Plasma Protein Analysis by Microparticle-based Proximity Ligation Assays
复制标题
DOI：
10.1074/mcp.m900248-mcp200
复制
发表时间：
2010-02-01
影响因子：
7
通讯作者：
Kamali-Moghaddam, Masood
中科院分区：
文献类型：
--
作者：
Darmanis, Spyros;Nong, Rachel Yuan;Kamali-Moghaddam, Masood
检测因疾病受损的组织释放到血流中的蛋白质可以促进病理状况的早期检测、鉴别诊断和治疗的随访。尽管有这些前景和大量的候选生物标志物，近年来建立新的蛋白质诊断检测方法的努力却取得了有限的成功。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测定，依赖于三个抗体分子同时识别靶蛋白以增加特异性。捕获微粒后，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品的同时检测和识别血液中的稀有蛋白质分子。我们证明，针对感兴趣的靶蛋白产生的单一多克隆抗体制剂可以很容易地用于建立检测，其中检测依赖于三个单独抗体分子的靶标识别，识别不同的表位。该测定法与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 的最先进的夹心 ELISA 进行了比较，发现它在动态范围和检测到的分子数量最少方面均优于该方法。此外，这些检测在未稀释的血浆和血清以及全血中表现出优异的性能，对九种不同的抗原产生了可比较的结果。因此，我们表明固相邻近连接测定适用于临床样品中广泛动态范围内的多种蛋白质生物标志物的验证。分子与细胞蛋白质组学 9：327-335，2010。
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. Molecular & Cellular Proteomics 9:327-335, 2010.