Sysmex Corporation, 4-4-4 Takatsukadai, Nishi-ku, Kobe 651-2271, Japan.
Anal Chem. 2016 Jul 19;88(14):7123-9. doi: 10.1021/acs.analchem.6b01148. Epub 2016 Jun 30.
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. This study presents a novel Digital ELISA that does not require droplets; instead, enzyme reaction products are concentrated using a tyramide signal amplification system. In our method, tyramide substrate reacts with horseradish peroxidase (HRP) labeled with an immunocomplex on beads, and the substrate is converted into short-lived radical intermediates. By adjusting the bead concentration in the HRP-tyramide reaction and conducting the reaction using freely moving beads, tyramide radicals are deposited only on beads labeled with HRP and there is no diffusion to other beads. Consequently, the fluorescence signal is localized on a portion of the beads, making it possible to count the number of labeled beads digitally. The performance of our method was demonstrated by detecting hepatitis B surface antigen with a limit of detection of 0.09 mIU/mL (139 aM) and a dynamic range of over 4 orders of magnitude. The obtained limit of detection represents a >20-fold higher sensitivity than conventional ELISA. Our method has potential applications in simple in vitro diagnostic systems for detecting ultralow concentrations of protein biomarkers.
数字酶联免疫吸附测定（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级液滴中。本研究提出了一种新颖的数字 ELISA，它不需要液滴；相反，使用辣根过氧化物酶（HRP）标记的免疫复合物的酪胺信号放大系统来浓缩酶反应产物。在我们的方法中，酪胺底物与珠上标记的辣根过氧化物酶（HRP）反应，底物转化为短寿命的自由基中间体。通过调整 HRP-酪胺反应中的珠浓度并使用自由移动的珠进行反应，只有标记有 HRP 的珠上沉积酪胺自由基，而不会扩散到其他珠上。因此，荧光信号定位于珠的一部分上，从而可以对标记的珠进行数字计数。我们的方法通过检测乙型肝炎表面抗原来证明其性能，检测限为 0.09 mIU/mL（139 aM），动态范围超过 4 个数量级。获得的检测限比传统 ELISA 高 20 多倍。我们的方法在用于检测超痕量蛋白生物标志物的简单体外诊断系统中有潜在的应用。
