Improved immunoassay sensitivity and specificity using single-molecule colocalization.
Improved immunoassay sensitivity and specificity using single-molecule colocalization.
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DOI：
10.1038/s41467-022-32796-x
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发表时间：
2022-09-12
影响因子：
16.6
通讯作者：
中科院分区：
文献类型：
--
作者：
酶联免疫吸附分析(ELISA)是现代分子检测的基石，但这项技术仍然面临着显著的挑战。最大的问题之一是区分目标分子产生的真实信号和非特定背景。在这里，我们开发了一种单分子共定位分析(SIMCA)，通过使用全内反射荧光显微镜来定量目标蛋白质，该方法基于来自正交标记捕获和检测抗体的荧光信号的共定位，从而克服了这一问题。通过对共定位信号的特异性计数，可以消除检测抗体非特异性结合产生的背景的影响。使用肿瘤坏死因子-α，我们表明，SIMCA的检测下限比传统的单色分析低三倍，并在血清和血液等复杂样本中表现出一致的分析性能。我们的结果有助于确定免疫分析中非特异性背景的有害影响，并展示通过消除这些影响可以实现的诊断收益。酶联免疫吸附试验的一个主要挑战是区分真实信号和非特异性结合。在这里，作者提出了一种单分子共定位分析(SIMCA)，它消除了这种影响，使皮摩尔蛋白浓度的可重复性检测成为可能。
Enzyme-linked immunosorbent assays (ELISAs) are a cornerstone of modern molecular detection, but the technique still faces notable challenges. One of the biggest problems is discriminating true signal generated by target molecules versus non-specific background. Here, we developed a Single-Molecule Colocalization Assay (SiMCA) that overcomes this problem by employing total internal reflection fluorescence microscopy to quantify target proteins based on the colocalization of fluorescent signal from orthogonally labeled capture and detection antibodies. By specifically counting colocalized signals, we can eliminate the effects of background produced by non-specific binding of detection antibodies. Using TNF-α, we show that SiMCA achieves a three-fold lower limit of detection compared to conventional single-color assays and exhibits consistent performance for assays performed in complex specimens such as serum and blood. Our results help define the pernicious effects of non-specific background in immunoassays and demonstrate the diagnostic gains that can be achieved by eliminating those effects. A major challenge of enzyme-linked immunosorbent assays is discriminating true signal from non-specific binding. Here the authors present a Single-Molecule Colocalization Assay (SiMCA) which eliminates such effects, enabling reproducible detection of picomolar protein concentrations.
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影响因子：
2.2
作者：
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通讯作者：
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影响因子：
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作者：
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DOI：
10.1016/j.jpba.2019.113010
发表时间：
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通讯作者：
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作者：
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通讯作者：
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