Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.
Nat Protoc. 2013 Jun;8(6):1234-48. doi: 10.1038/nprot.2013.070. Epub 2013 May 30.
Solid-phase proximity ligation assays share properties with the classical sandwich immunoassays for protein detection. The proteins captured via antibodies on solid supports are, however, detected not by single antibodies with detectable functions, but by pairs of antibodies with attached DNA strands. Upon recognition by these sets of three antibodies, pairs of DNA strands brought in proximity are joined by ligation. The ligated reporter DNA strands are then detected via methods such as real-time PCR or next-generation sequencing (NGS). We describe how to construct assays that can offer improved detection specificity by virtue of recognition by three antibodies, as well as enhanced sensitivity owing to reduced background and amplified detection. Finally, we also illustrate how the assays can be applied for parallel detection of proteins, taking advantage of the oligonucleotide ligation step to avoid background problems that might arise with multiplexing. The protocol for the singleplex solid-phase proximity ligation assay takes ~5 h. The multiplex version of the assay takes 7-8 h depending on whether quantitative PCR (qPCR) or sequencing is used as the readout. The time for the sequencing-based protocol includes the library preparation but not the actual sequencing, as times may vary based on the choice of sequencing platform.
固相邻近连接分析与经典的蛋白质检测夹心免疫分析具有相同的性质。然而，固相上捕获的蛋白质不是通过具有可检测功能的单克隆抗体检测，而是通过带有附着 DNA 链的成对抗体检测。在这些三抗体识别后，通过连接将靠近的 DNA 链连接起来。然后通过实时 PCR 或下一代测序 (NGS) 等方法检测连接的报告 DNA 链。我们描述了如何构建可以通过三抗体识别提高检测特异性的分析，以及通过减少背景和放大检测提高灵敏度的分析。最后，我们还说明了如何利用寡核苷酸连接步骤来避免多重化可能出现的背景问题，从而实现蛋白质的平行检测。单重固相邻近连接分析的方案耗时约 5 小时。根据使用定量 PCR(qPCR) 还是测序作为读出方法，多重分析的方案耗时 7-8 小时。基于测序的方案时间包括文库制备，但不包括实际测序，因为测序平台的选择可能会导致时间不同。
