Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland 21218, United States.
Department of Mechanical Engineering, Johns Hopkins University, Baltimore, Maryland 21218, United States.
Anal Chem. 2022 Sep 13;94(36):12481-12489. doi: 10.1021/acs.analchem.2c02684. Epub 2022 Aug 30.
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.
许多蛋白质生物标志物在生物体液中以非常低的水平存在，但可能在重要的生物学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，这凸显了开发用于高度敏感蛋白质检测的新工具的重要性。尽管数字酶联免疫吸附测定（ELISA）与传统 ELISA 相比表现出超高的灵敏度，但专用仪器的要求限制了其可及性并阻止了其广泛实施。另一方面，邻近连接测定（PLA）和邻近延伸测定（PEA）使用常规实验室设备进行灵敏和特异性蛋白质检测，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，我们开发了一种基于磁性珠的 PEA（magPEA），它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、均相 PEA 和固相 PLA 相比，将磁性珠纳入 PEA 工作流程可实现数量级的灵敏度提高，并在使用 IL-6、IL-8 和 GM-CSF 进行验证时达到接近数字 ELISA 的检测限。我们的 magPEA 提供了一种简单的高灵敏度蛋白质检测方法，可轻松在其他实验室中实施，因此最终将加速未来对低丰度蛋白质生物标志物的研究。
