Highly sensitive multiplex protein detection by droplet-free digital ELISA
Highly sensitive multiplex protein detection by droplet-free digital ELISA
复制标题
DOI：
10.1002/ecj.12137
复制
发表时间：
2019-02-01
影响因子：
0.3
通讯作者：
Suzuki, Seigo
中科院分区：
文献类型：
--
作者：
Akama, Kenji;Shirai, Kentaro;Suzuki, Seigo
数字化酶联免疫吸附试验（ELISA）是一种单分子计数技术，是最灵敏的免疫分析方法之一。该技术的关键方面是将来自单个靶分子的酶反应产物浓缩在飞升液滴中。另一方面，我们开发了一种新的数字ELISA，不需要液滴;相反，酶反应产物使用酪胺信号放大系统浓缩。在这项研究中，我们提出了一种方法，使蛋白质的多重检测的基础上无液滴数字ELISA。首先，用样品溶液孵育固定有对不同靶蛋白特异性的抗体的两种类型的顺磁珠，并将靶蛋白捕获在其特异性珠上。然后，用辣根过氧化物酶（HRP）标记这些珠子，酪胺底物与珠子上的HRP反应，导致该反应的产物沉积在这些珠子上。珠上的这种信号放大使得可以对标记的珠的数量进行数字计数。我们用这种方法同时检测IL-6和HBsAg的单分子分辨率。方法的检出限分别为0.1 pg/mL和0.013 IU/mL。我们的方法在简单的体外诊断系统中具有潜在的应用，可以同时和高灵敏度地检测蛋白质生物标志物。
Digital enzyme-linked immunosorbent assay (ELISA) is a single molecule counting technology and is one of the most sensitive immunoassay methods. The key aspect of this technology is to concentrate enzyme reaction products from a single target molecule in femtoliter droplets. On the other hand, we have developed a novel Digital ELISA that does not require droplets; instead, enzyme reaction products are concentrated using a tyramide signal amplification system. In this study, we present a method that enables the multiplex detection of proteins based on Droplet-free Digital ELISA. First, two types of paramagnetic beads immobilized with antibodies specific to different target proteins were incubated with a sample solution, and target proteins were captured on their specific beads. Then, these beads were labeled with horseradish peroxidase (HRP), and tyramide substrate reacted with HRP on beads, resulting in products of this reaction deposited on those beads. This signal amplification on beads makes it possible to count the number of labeled beads digitally. We used this approach to simultaneously detect IL-6 and HBs Ag with single molecule resolution. The obtained limit of detections were 0.1 pg/mL and 0.013 IU/mL, respectively. Our method has potential applications in simple in vitro diagnostic systems for simultaneous and high-sensitive detection of protein biomarkers.