title: Solid-phase proximity ligation assays for individual or parallel protein analyses with readout via real-time PCR or sequencing.
abstract: 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.
authors.affiliation: Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.
authors.forename: Rachel Yuan
authors.initials: RY
authors.lastname: Nong
authors.forename: Di
authors.initials: D
authors.lastname: Wu
authors.forename: Junhong
authors.initials: J
authors.lastname: Yan
authors.forename: Maria
authors.initials: M
authors.lastname: Hammond
authors.forename: Gucci Jijuan
authors.initials: GJ
authors.lastname: Gu
authors.forename: Masood
authors.initials: M
authors.lastname: Kamali-Moghaddam
authors.forename: Ulf
authors.initials: U
authors.lastname: Landegren
authors.forename: Spyros
authors.initials: S
authors.lastname: Darmanis
mesh_terms: D000906:Antibodies / Q000378:metabolism*; D007118:Immunoassay / Q000379:methods*; D009841:Oligonucleotides / Q000235:genetics; D011506:Proteins / Q000032:analysis* / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA / Q000379:methods
chemical_list: D000906:Antibodies; D009841:Oligonucleotides; D011506:Proteins