Stanford Genome Technology Center, Stanford University School of Medicine, Palo Alto, CA 94304.
Department of Chemical Engineering, Stanford University, Stanford, CA 94305.
Proc Natl Acad Sci U S A. 2018 Jan 30;115(5):E925-E933. doi: 10.1073/pnas.1718283115. Epub 2018 Jan 16.
Proximity ligation assay (PLA) is a powerful tool for quantitative detection of protein biomarkers in biological fluids and tissues. Here, we present the circular proximity ligation assay (c-PLA), a highly specific protein detection method that outperforms traditional PLA in stringency, ease of use, and compatibility with low-affinity reagents. In c-PLA, two proximity probes bind to an analyte, providing a scaffolding that positions two free oligonucleotides such that they can be ligated into a circular DNA molecule. This assay format stabilizes antigen proximity probe complexes and enhances stringency by reducing the probability of random background ligation events. Circle formation also increases selectivity, since the uncircularized DNA can be removed enzymatically. We compare this method with traditional PLA on several biomarkers and show that the higher stringency for c-PLA improves reproducibility and enhances sensitivity in both buffer and human plasma. The limit of detection ranges from femtomolar to nanomolar concentrations for both methods. Kinetic analyses using surface plasmon resonance (SPR) and biolayer interferometry (BLI) reveal that the variation in limit of detection is due to the variation in antibody affinity and that c-PLA outperforms traditional PLA for low-affinity antibodies. The lower background signal can be used to increase proximity probe concentration while maintaining a high signal-to-noise ratio, thereby enabling the use of low-affinity reagents in a homogeneous assay format. We anticipate that the advantages of c-PLA will be useful in a variety of clinical protein detection applications where high-affinity reagents are lacking.
邻近连接分析（PLA）是一种强大的工具，可用于定量检测生物体液和组织中的蛋白质生物标志物。在这里，我们介绍了圆形邻近连接分析（c-PLA），这是一种高度特异性的蛋白质检测方法，在严格性、易用性和与低亲和力试剂的兼容性方面优于传统的 PLA。在 c-PLA 中，两个邻近探针与分析物结合，提供一个支架，将两个游离寡核苷酸定位，以便它们可以连接成一个圆形 DNA 分子。这种检测格式稳定了抗原邻近探针复合物，并通过减少随机背景连接事件的概率来提高严格性。圆形形成还增加了选择性，因为未环化的 DNA 可以通过酶去除。我们在几个生物标志物上比较了这种方法与传统的 PLA，并表明 c-PLA 的更高严格性提高了缓冲液和人血浆中的重现性和灵敏度。两种方法的检测限范围从飞摩尔到纳摩尔浓度。使用表面等离子体共振（SPR）和生物层干涉（BLI）的动力学分析表明，检测限的变化是由于抗体亲和力的变化，并且 c-PLA 对低亲和力抗体的性能优于传统的 PLA。较低的背景信号可用于增加邻近探针浓度，同时保持高信噪比，从而能够在均相检测格式中使用低亲和力试剂。我们预计 c-PLA 的优势将在各种缺乏高亲和力试剂的临床蛋白质检测应用中有用。
