Highly Sensitive Serum Protein Analysis Using Magnetic Bead-Based Proximity Extension Assay.
Highly Sensitive Serum Protein Analysis Using Magnetic Bead-Based Proximity Extension Assay.
复制标题
使用基于磁珠的邻近延伸分析进行高灵敏度血清蛋白分析。
DOI：
10.1021/acs.analchem.2c02684
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发表时间：
2022
影响因子：
7.4
通讯作者：
Wang,Tza-Huei
中科院分区：
文献类型：
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作者：
Zhang,Pengfei;Hu,Jiumei;Park,JoonSoo;Hsieh,Kuangwen;Chen,Liben;Mao,Alan;Wang,Tza-Huei
许多蛋白质生物标志物以非常低的水平存在于生物流体中，但可能在重要的生物过程中发挥关键作用。这些低丰度蛋白质在很大程度上尚未被探索，这一事实强调了开发高灵敏度蛋白质检测新工具的重要性。尽管数字化酶联免疫吸附测定（ELISA）与传统ELISA相比具有更高的灵敏度，但对专用仪器的要求限制了其可及性并阻碍了其广泛实施。另一方面，邻近连接测定（PLA）和邻近延伸测定（PEA）使用常规实验室设置显示出灵敏和特异性的蛋白质检测，但它们的灵敏度需要进一步提高以匹配数字ELISA。为了实现具有最小可及性限制的高灵敏度蛋白质检测，我们开发了基于磁珠的PEA（magPEA），其具有三重表位识别要求，并能够进行广泛的洗涤以提高灵敏度和增强特异性。我们证明，与传统ELISA、均质PEA和固相PLA相比，将磁珠并入PEA工作流程有助于灵敏度提高几个数量级，并且当使用IL-6、IL-8和GM-CSF作为验证时，实现了接近数字ELISA的检测限。我们的magPEA为高灵敏度蛋白质检测提供了一种简单的方法，可以很容易地应用于其他实验室，从而最终加速未来低丰度蛋白质生物标志物的研究。
Many protein biomarkers are present in biofluids at a very low level but may play critical roles in important biological processes. The fact that these low-abundance proteins remain largely unexplored underscores the importance of developing new tools for highly sensitive protein detection. Although digital enzyme-linked immunosorbent assay (ELISA) has demonstrated ultrahigh sensitivity compared with conventional ELISA, the requirement of specialized instruments limits the accessibility and prevents the widespread implementation. On the other hand, proximity ligation assays (PLA) and proximity extension assays (PEA) show sensitive and specific protein detection using regular laboratory setups, but their sensitivity needs to be further improved to match digital ELISA. To achieve highly sensitive protein detection with minimal accessibility limitation, we develop a magnetic bead-based PEA (magPEA), which posts triple epitope recognition requirement and enables extensive washing for improved sensitivity and enhanced specificity. We demonstrate that the incorporation of magnetic beads into PEA workflow facilitates orders of magnitude sensitivity improvement compared with conventional ELISA, homogeneous PEA, and solid-phase PLA and achieves limits of detection close to that of digital ELISA when using IL-6, IL-8, and GM-CSF as validation. Our magPEA provides a simple approach for highly sensitive protein detection that can be readily implemented to other laboratories and will thus ultimately accelerate the study of the low abundance protein biomarkers in the future.