1. Streamlined circular proximity ligation assay provides high stringency and compatibility with low-affinity antibodies.
- Author
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Jalili R, Horecka J, Swartz JR, Davis RW, and Persson HHJ
- Subjects
- Antibody Affinity, DNA, Single-Stranded chemistry, Dose-Response Relationship, Drug, Humans, Immunoassay, Oligonucleotides, Phosphorylation, Polymerase Chain Reaction, Protein Binding, Proteomics, Reproducibility of Results, Antibodies chemistry, Biomarkers chemistry, Blood Proteins chemistry, Protein Interaction Mapping methods
- Abstract
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., Competing Interests: Conflict of interest statement: S.F. owns stock in a company (Olink AB) with patents on the core technology described, "Proximity Ligation Assay."
- Published
- 2018
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