{ "id": "epg2q2qd1n", "slug": "proximity-extension-assay", "title": "Proximity extension assay", "updated": "2026-10-25", "topic_path": [ { "id": "life", "label": "Life and health", "api_url": "https://www.edgepedia.org/api/v1/topics/life" }, { "id": "life.health", "label": "Human health and medicine", "api_url": "https://www.edgepedia.org/api/v1/topics/life.health" }, { "id": "life.health.clinical", "label": "Clinical assessment and procedures", "api_url": "https://www.edgepedia.org/api/v1/topics/life.health.clinical" }, { "id": "life.health.clinical.laboratory-assays-and-specimen-processing", "label": "Laboratory assays and specimen processing", "api_url": "https://www.edgepedia.org/api/v1/topics/life.health.clinical.laboratory-assays-and-specimen-processing" } ], "geo": [ { "id": "geo.nongeo.t2001.life.health.clinical", "label": "Non-geographic · 2001 to 2020: Clinical assessment and procedures", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t2001.life.health.clinical", "path": [ { "id": "geo.nongeo", "label": "Non-geographic", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo" }, { "id": "geo.nongeo.t2001", "label": "Non-geographic · 2001 to 2020", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t2001" }, { "id": "geo.nongeo.t2001.life", "label": "Life and health", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t2001.life" }, { "id": "geo.nongeo.t2001.life.health", "label": "Human health and medicine", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t2001.life.health" }, { "id": "geo.nongeo.t2001.life.health.clinical", "label": "Clinical assessment and procedures", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t2001.life.health.clinical" } ] } ], "excerpt": "The proximity extension assay (PEA) is a homogeneous immunoassay, introduced in 2011, in which pairs of DNA-linked antibodies quantify thousands of proteins from microliter plasma samples by PCR or sequencing.", "snippet": "The proximity extension assay (PEA) is a homogeneous immunoassay, introduced in 2011, in which pairs of DNA-linked antibodies quantify thousands of proteins from microliter plasma samples by PCR or sequencing.", "node": "life.health.clinical.laboratory-assays-and-specimen-processing", "markdown": "# Proximity extension assay\n\nThe proximity extension assay (PEA) is a homogeneous immunoassay in which pairs of antibodies carrying DNA oligonucleotides generate a PCR-amplifiable template only when both bind the same target protein, allowing dozens to thousands of proteins to be quantified from microliter samples by qPCR or next-generation sequencing (NGS).[1](https://europepmc.org/articles/PMC3159481) • [2](https://olink.com/technology/what-is-pea) It is used for biomarker discovery and validation in plasma, serum, cerebrospinal fluid (CSF), and other specimen types, and underlies population-scale plasma proteomics such as the UK Biobank Pharma Proteomics Project.[3](https://info.olink.com/hubfs/000-documents/05-white%20paper/1328-Olink-White-paper-Olink-technology-and-mass%20spectrometry.pdf?hsLang=en)\n\n| Key fact | Detail |\n|---|---|\n| Output | Relative protein levels as NPX (Normalized Protein eXpression) values on a \\( \\log_{2} \\) scale, from qPCR Cq values or sequencing counts; absolute pg/mL only on certain panels[4](https://7074596.fs1.hubspotusercontent-na1.net/hubfs/7074596/01-User%20Manuals%20for%20website/1415-olink-target-96-user-manual-fast.pdf) • [5](https://www.nature.com/articles/s41467-021-22767-z) • [3](https://info.olink.com/hubfs/000-documents/05-white%20paper/1328-Olink-White-paper-Olink-technology-and-mass%20spectrometry.pdf?hsLang=en) |\n| Multiplexing | 92-plex (Target 96), nearly 3000 proteins (Explore 3072), about 5400 proteins (Explore HT)[4](https://7074596.fs1.hubspotusercontent-na1.net/hubfs/7074596/01-User%20Manuals%20for%20website/1415-olink-target-96-user-manual-fast.pdf) • [3](https://info.olink.com/hubfs/000-documents/05-white%20paper/1328-Olink-White-paper-Olink-technology-and-mass%20spectrometry.pdf?hsLang=en) • [6](https://7074596.fs1.hubspotusercontent-na1.net/hubfs/7074596/04-Validation%20data/1345-Olink%20Explore-HT-Validation-Data.pdf) |\n| Sample volume | 1 µL (Target 96), 2 µL (Explore HT), 2.8 µL in the original Explore protocol[4](https://7074596.fs1.hubspotusercontent-na1.net/hubfs/7074596/01-User%20Manuals%20for%20website/1415-olink-target-96-user-manual-fast.pdf) • [6](https://7074596.fs1.hubspotusercontent-na1.net/hubfs/7074596/04-Validation%20data/1345-Olink%20Explore-HT-Validation-Data.pdf) • [5](https://www.nature.com/articles/s41467-021-22767-z) |\n| Specificity | 99.8% of 1472 Explore assays and 99.5% of 5416 Explore HT assays showed no cross-reactivity in validation testing[7](https://pmc.ncbi.nlm.nih.gov/articles/PMC8633680/) • [6](https://7074596.fs1.hubspotusercontent-na1.net/hubfs/7074596/04-Validation%20data/1345-Olink%20Explore-HT-Validation-Data.pdf) |\n| Dynamic range | Over 10 orders of magnitude across assays (fg/mL to mg/mL) on Explore HT; average per-assay quantifiable range 2.7 \\( \\log_{10} \\) (~500-fold) on Explore[6](https://7074596.fs1.hubspotusercontent-na1.net/hubfs/7074596/04-Validation%20data/1345-Olink%20Explore-HT-Validation-Data.pdf) • [7](https://pmc.ncbi.nlm.nih.gov/articles/PMC8633680/) |\n| Throughput | Explore HT: over 5400 proteins in 344 samples per NGS run; UKB-PPP profiled up to 2923 proteins in 55,000 participants[3](https://info.olink.com/hubfs/000-documents/05-white%20paper/1328-Olink-White-paper-Olink-technology-and-mass%20spectrometry.pdf?hsLang=en) |\n| Introduced | Martin Lundberg and colleagues, Nucleic Acids Research, 2011[8](https://doi.org/10.1093/nar/gkr424) |\n\n## How it works\n\nEach target protein is recognized by two antibodies, each covalently linked to a DNA strand. In the original design, the antibodies carried 40-mer oligonucleotides (one attached at the 3'-end, one at the 5'-end) containing a 20-bp universal hybridization site and a unique 20-bp qPCR primer site; a 56-mer oligonucleotide hybridizes to the 3'-linked probe and is extended.[1](https://europepmc.org/articles/PMC3159481) When both antibodies bind the same protein, their oligonucleotides are brought close enough to hybridize, and a [DNA polymerase](https://www.edgepedia.org/dna-polymerase) extends one strand to create a double-stranded barcode that is unique to that target and quantified by PCR or sequencing.[2](https://olink.com/technology/what-is-pea) Requiring two probes to be in proximity greatly reduces signal from unbound or nonspecifically bound probes, but it does not eliminate background, since random proximity and cross-reactivity can still produce signal even in highly multiplexed mixtures.[2](https://olink.com/technology/what-is-pea)\n\nPolymerase choice matters for background: 3'-exonuclease-capable polymerases (for example T4 DNA polymerase) proved superior in sensitivity because they degrade non-proximal DNA strands and thereby suppress background.[1](https://europepmc.org/articles/PMC3159481) When multiplexing was scaled from 24- to 96-plex, a third specificity layer was added: each probe pair received a unique 5-base-pair annealing site that prevents non-matching probes from binding each other, addressing proximity effects between probes that have cross-reactively bound abundant proteins.[9](https://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0095192)\n\n## How it is done\n\nThe practitioner workflow, as implemented in the 96-plex Olink Target 96 format, has three core steps.[4](https://7074596.fs1.hubspotusercontent-na1.net/hubfs/7074596/01-User%20Manuals%20for%20website/1415-olink-target-96-user-manual-fast.pdf)\n\n1. **Incubation.** 1 µL of sample is incubated overnight (16–22 h) with the paired probe mixes.[4](https://7074596.fs1.hubspotusercontent-na1.net/hubfs/7074596/01-User%20Manuals%20for%20website/1415-olink-target-96-user-manual-fast.pdf) In the 2014 protocol this was at 4 °C; extension then runs 20 min at 50 °C with a hyper-thermostable Pwo polymerase system that allows room-temperature reagent addition.[9](https://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0095192)\n2. **Extension and preamplification.** Proximal probes are extended and the resulting barcodes amplified, classically through 17 preamplification cycles with universal primers.[9](https://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0095192)\n3. **Detection.** Amplicons are read by microfluidic qPCR (a Fluidigm 96.96 Dynamic Array in the 2014 protocol, about 4.5 h)[9](https://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0095192) • [4](https://7074596.fs1.hubspotusercontent-na1.net/hubfs/7074596/01-User%20Manuals%20for%20website/1415-olink-target-96-user-manual-fast.pdf) or, in NGS-based formats, sequenced; only reads with exact barcode matches are approved.[7](https://pmc.ncbi.nlm.nih.gov/articles/PMC8633680/)\n\n[Quality control](https://www.edgepedia.org/quality-control) uses internal controls spiked into every sample: two Incubation Controls (non-human antigens), an Extension Control (an antibody with DNA tags permanently in proximity), and a Detection Control (a complete double-stranded amplicon), monitoring the immuno reaction, extension, and amplification/detection steps respectively.[4](https://7074596.fs1.hubspotusercontent-na1.net/hubfs/7074596/01-User%20Manuals%20for%20website/1415-olink-target-96-user-manual-fast.pdf) An Inter-plate Control, a pool of 92 probe pairs held in fixed proximity run in triplicate, normalizes assays between plates.[4](https://7074596.fs1.hubspotusercontent-na1.net/hubfs/7074596/01-User%20Manuals%20for%20website/1415-olink-target-96-user-manual-fast.pdf) Results are reported as NPX values, a relative \\( \\log_{2} \\)-scale unit derived from Cq values; equal NPX values for two proteins do not mean equal concentrations, and NPX values must not be compared between runs without inter-plate normalization.[4](https://7074596.fs1.hubspotusercontent-na1.net/hubfs/7074596/01-User%20Manuals%20for%20website/1415-olink-target-96-user-manual-fast.pdf)\n\n## Origin\n\nPEA was introduced by Martin Lundberg and colleagues in a 2011 Nucleic Acids Research paper demonstrating sensitive, specific detection of low-abundant proteins in human blood.[8](https://doi.org/10.1093/nar/gkr424) It grew out of the [proximity ligation assay](https://www.edgepedia.org/proximity-ligation-assay) (PLA), reported by Simon Fredriksson and colleagues in [Nature Biotechnology](https://www.edgepedia.org/nature-biotechnology) in 2002, in which proximal DNA probes are joined by ligation rather than polymerization.[10](https://doi.org/10.1038/nbt0502-473) The 2011 group replaced the [DNA ligase](https://www.edgepedia.org/dna-ligase) with DNA polymerases because ligase-based proximity probes suffered recovery loss in complex fluids such as blood plasma, where polymerases proved less prone to enzymatic inhibition.[8](https://doi.org/10.1093/nar/gkr424) Subsequent development produced a homogeneous 96-plex immunoassay reported by Erika Assarsson, Martin Lundberg, and colleagues in 2014,[11](https://doi.org/10.1371/journal.pone.0095192) the combination of PEA with NGS readout reported by Lotta Wik and colleagues in 2021,[12](https://doi.org/10.1016/j.mcpro.2021.100168) and a quantitative 21-protein cardiovascular-risk panel (CVD-21) reported by Agneta Siegbahn and colleagues in 2023.[13](https://doi.org/10.1371/journal.pone.0293465)\n\n## Variants\n\nThe commercial portfolio spans qPCR-based and sequencing-based formats. Olink Target 96 panels assay 92 biomarkers from 1 µL with results in 24 hours; Olink Flex runs individually tailored studies combining up to 30 biomarkers from a library of about 200 pre-validated assays; Olink Focus covers up to 21 pre-validated assays.[4](https://7074596.fs1.hubspotusercontent-na1.net/hubfs/7074596/01-User%20Manuals%20for%20website/1415-olink-target-96-user-manual-fast.pdf) • [3](https://info.olink.com/hubfs/000-documents/05-white%20paper/1328-Olink-White-paper-Olink-technology-and-mass%20spectrometry.pdf?hsLang=en) Olink Explore 3072 measures nearly 3000 proteins in 352 samples per NGS run, and Olink Explore HT measures over 5400 proteins in 344 samples from 2 µL.[3](https://info.olink.com/hubfs/000-documents/05-white%20paper/1328-Olink-White-paper-Olink-technology-and-mass%20spectrometry.pdf?hsLang=en) • [6](https://7074596.fs1.hubspotusercontent-na1.net/hubfs/7074596/04-Validation%20data/1345-Olink%20Explore-HT-Validation-Data.pdf) The Signature Q100 is a benchtop instrument format.[2](https://olink.com/technology/what-is-pea) At population scale, the UK Biobank Pharma Proteomics Project, funded by a consortium of thirteen biopharmaceutical companies, profiled up to 2923 unique assays per sample in 55,000 participant samples analyzed between April 2021 and February 2022.[14](https://biobank.ctsu.ox.ac.uk/crystal/ukb/docs/Olink_proteomics_data.pdf)\n\n## Applications\n\nPEA is applied to plasma, serum, CSF, saliva, urine, dried blood spots, cell and tissue lysate, interstitial fluid, breast milk, synovial fluid, exosomes, and fine-needle biopsies.[7](https://pmc.ncbi.nlm.nih.gov/articles/PMC8633680/) A 2025 Methods in Molecular Biology chapter states the technology has been applied in more than 2,000 peer-reviewed studies of biomarker discovery in diagnostics, prognostics, and personalized medicine.[15](https://europepmc.org/article/med/40601148) Clinical validation examples include the CVD-21 cardiovascular panel[13](https://doi.org/10.1371/journal.pone.0293465) and a 2026 study of the Olink Target 48 Neurodegeneration panel in 238 dementia patients and 65 controls, in which plasma pTau217 achieved an AUC exceeding 0.91 for Alzheimer's disease diagnosis.[16](https://springerlink.fh-diploma.de/article/10.1186/s13195-026-02142-0)\n\nPre-analytical factors matter. Panels are validated on EDTA plasma and serum; citrate plasma, heparin plasma, tissue and cell lysates, CSF, and saliva are also compatible, but formalin-fixed paraffin-embedded tissues are not, and extreme IgG levels or fluorescent particles can interfere.[4](https://7074596.fs1.hubspotusercontent-na1.net/hubfs/7074596/01-User%20Manuals%20for%20website/1415-olink-target-96-user-manual-fast.pdf) • [3](https://info.olink.com/hubfs/000-documents/05-white%20paper/1328-Olink-White-paper-Olink-technology-and-mass%20spectrometry.pdf?hsLang=en) Serum and EDTA plasma results cannot be directly compared: on a 92-plex immuno-oncology panel, 36 of 80 evaluable proteins failed one or more serum-to-EDTA-plasma reproducibility criteria, with mean serum-to-EDTA-plasma ratios ranging 0.41 to 3.01.[17](https://clinicalproteomicsjournal.biomedcentral.com/counter/pdf/10.1186/s12014-022-09380-y.pdf)\n\n## Limitations and alternatives\n\n**Quantification is relative.** NPX values are relative units on a [logarithmic scale](https://www.edgepedia.org/logarithmic-scale), not absolute concentrations, and cannot be compared across plates without bridging-sample normalization; in one six-study analysis, mean inter-study CV across 81 proteins fell from 41.3% before normalization to 26.2% after bridging.[17](https://clinicalproteomicsjournal.biomedcentral.com/counter/pdf/10.1186/s12014-022-09380-y.pdf) A substantial fraction of assays return values below the limit of detection, especially for low-abundance proteins, and this fraction correlates with CV (Spearman 0.69).[18](https://www.nature.com/articles/s41586-023-06563-x) The high-dose hook effect limits the upper end of each assay's quantifiable range.[7](https://pmc.ncbi.nlm.nih.gov/articles/PMC8633680/) Interference testing on the 96-plex panel showed no effect of bilirubin up to 300 µg/mL or intralipid up to 10 mg/mL, but 7 of 92 assays increased significantly with 15 g/L hemolysate, attributed to analytes leaking from lysed cells.[9](https://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0095192)\n\n**Platform comparisons.** Sensitivity of the 96-plex assays was comparable to corresponding R&D Systems ELISAs, with roughly half of assays showing similar or better sensitivity.[9](https://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0095192) Against the aptamer-based SomaScan, a [UK Biobank](https://www.edgepedia.org/uk-biobank)-scale comparison found a similar absolute number of cis-pQTL-supported assays (2,101 Olink vs 2,120 SomaScan) but a higher proportion of supported assays on Olink (72% vs 43%), while median duplicate CV was higher for Olink (16.5% vs 9.9%).[18](https://www.nature.com/articles/s41586-023-06563-x) The manufacturer states precision of <10% intra-assay and <20% inter-assay CV,[3](https://info.olink.com/hubfs/000-documents/05-white%20paper/1328-Olink-White-paper-Olink-technology-and-mass%20spectrometry.pdf?hsLang=en) and the ARIC study found a median inter-assay CV of 3.4% for Olink proteins,[19](https://pmc.ncbi.nlm.nih.gov/articles/PMC9812856/) so realized precision depends on cohort and assay. In ARIC, the average Spearman correlation between SomaScan v4 and Olink Proseek across 417 protein comparisons was \\( r = 0.46 \\), with only 19% of comparisons at \\( r \\geq 0.8 \\).[19](https://pmc.ncbi.nlm.nih.gov/articles/PMC9812856/) Both platforms are susceptible to epitope effects: about 23% of sentinel Olink and 24% of sentinel SomaScan cis pQTLs in high linkage disequilibrium with protein-altering variants are likely caused by antibodies or aptamers binding the altered epitope rather than true protein-level variation.[18](https://www.nature.com/articles/s41586-023-06563-x) Published sources do not give cost figures for PEA versus ELISA, Simoa, or MSD, nor a direct PEA-versus-Simoa sensitivity benchmark.\n\n## References\n\n1. [Homogeneous antibody-based proximity extension assays provide sensitive and specific detection of low-abundant proteins in human blood (Lundberg et al., Nucleic Acids Research, 2011)](https://europepmc.org/articles/PMC3159481)\n2. [What is PEA?, Olink®](https://olink.com/technology/what-is-pea)\n3. [How Olink technology complements mass spectrometry (Olink white paper)](https://info.olink.com/hubfs/000-documents/05-white%20paper/1328-Olink-White-paper-Olink-technology-and-mass%20spectrometry.pdf?hsLang=en)\n4. [Olink® Target 96 User Manual](https://7074596.fs1.hubspotusercontent-na1.net/hubfs/7074596/01-User%20Manuals%20for%20website/1415-olink-target-96-user-manual-fast.pdf)\n5. [Next generation plasma proteome profiling to monitor health and disease (Nature Communications, 2021)](https://www.nature.com/articles/s41467-021-22767-z)\n6. [Olink Explore HT Validation Data](https://7074596.fs1.hubspotusercontent-na1.net/hubfs/7074596/04-Validation%20data/1345-Olink%20Explore-HT-Validation-Data.pdf)\n7. [Proximity Extension Assay in Combination with Next-Generation Sequencing for High-throughput Proteome-wide Analysis (Wik et al., Mol Cell Proteomics, 2021)](https://pmc.ncbi.nlm.nih.gov/articles/PMC8633680/)\n8. [Martin Lundberg and colleagues (2011). Homogeneous antibody-based proximity extension assays provide sensitive and specific detection of low-abundant proteins in human blood. Nucleic Acids Research.](https://doi.org/10.1093/nar/gkr424)\n9. [Homogenous 96-Plex PEA Immunoassay Exhibiting High Sensitivity, Specificity, and Excellent Scalability (Assarsson et al., PLOS ONE, 2014)](https://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0095192)\n10. [Simon Fredriksson and colleagues (2002). Protein detection using proximity-dependent DNA ligation assays. Nature Biotechnology.](https://doi.org/10.1038/nbt0502-473)\n11. [Erika Assarsson and colleagues (2014). Homogenous 96-Plex PEA Immunoassay Exhibiting High Sensitivity, Specificity, and Excellent Scalability. PLoS ONE.](https://doi.org/10.1371/journal.pone.0095192)\n12. [Lotta Wik and colleagues (2021). Proximity Extension Assay in Combination with Next-Generation Sequencing for High-throughput Proteome-wide Analysis. Molecular & Cellular Proteomics.](https://doi.org/10.1016/j.mcpro.2021.100168)\n13. [Agneta Siegbahn and colleagues (2023). Development and validation of a quantitative Proximity Extension Assay instrument with 21 proteins associated with cardiovascular risk (CVD-21). PLoS ONE.](https://doi.org/10.1371/journal.pone.0293465)\n14. [UK Biobank Olink proteomics data (version 1.0, March 2023)](https://biobank.ctsu.ox.ac.uk/crystal/ukb/docs/Olink_proteomics_data.pdf)\n15. [Proteomics by qPCR Using the Proximity Extension Assay (PEA). Methods in Molecular Biology, 2025;2929:129-142](https://europepmc.org/article/med/40601148)\n16. [Analytical and clinical validation of a novel proximity extension assay-based plasma biomarker panel in a cohort of prevalent neurodegenerative dementias (Alzheimer's Research & Therapy, 2026)](https://springerlink.fh-diploma.de/article/10.1186/s13195-026-02142-0)\n17. [Determination of temporal reproducibility and variability of cancer biomarkers in serum and EDTA plasma samples using a proximity extension assay (Clinical Proteomics)](https://clinicalproteomicsjournal.biomedcentral.com/counter/pdf/10.1186/s12014-022-09380-y.pdf)\n18. [Large-scale plasma proteomics comparisons through genetics and disease associations (Nature, 2023)](https://www.nature.com/articles/s41586-023-06563-x)\n19. [Comparison of proteomic measurements across platforms in the ARIC Study](https://pmc.ncbi.nlm.nih.gov/articles/PMC9812856/)\n\n---\n*Topic: Encyclopedia › Life and health › Human health and medicine › Clinical assessment and procedures › Laboratory assays and specimen processing*\n\n*Initially written Sep 29, 2026 · Edited: Oct 15, 2026; Oct 21, 2026; Oct 25, 2026*\n\n*Copyright 2026 EdgeChat AI, a subsidiary of Biostate AI.*\n\nLicense: Edgepedia Community License 1.0, https://www.edgepedia.org/edgepedia/license\n", "same_as": [], "url": "https://www.edgepedia.org/proximity-extension-assay", "markdown_url": "https://www.edgepedia.org/proximity-extension-assay.md", "license": { "name": "Edgepedia Community License 1.0", "url": "https://www.edgepedia.org/edgepedia/license", "summary": "Free with credit, commercial use included. AI training is open to everyone. For other uses, organizations over USD 100M in revenue or 100M monthly users license separately.", "spdx": "LicenseRef-Edgepedia-Community-1.0" }, "credit": "\"Proximity extension assay\", Edgepedia (EdgeChat), https://www.edgepedia.org/proximity-extension-assay. Edgepedia Community License 1.0.", "credit_md": "\"[Proximity extension assay](https://www.edgepedia.org/proximity-extension-assay)\", Edgepedia (EdgeChat), [https://www.edgepedia.org/proximity-extension-assay](https://www.edgepedia.org/proximity-extension-assay). [Edgepedia Community License 1.0](https://www.edgepedia.org/edgepedia/license).", "credit_html": "\"Proximity extension assay\", Edgepedia (EdgeChat), https://www.edgepedia.org/proximity-extension-assay. Edgepedia Community License 1.0.", "speakable": "The proximity extension assay is a homogeneous immunoassay, introduced in 2011, in which pairs of DNA-linked antibodies quantify thousands of proteins from microliter plasma samples by PCR or sequencing." }