59:. Target binding by the proximity probes increases their local relative effective concentration of the DNA-tags enabling hybridization of weak complementarity to each other which then enables a DNA polymerase mediated extension forming a united DNA sequence specific for each target protein detected. The use of 3'exonuclease proficient polymerases lowers background noise and hyper thermostable polymerases mediate a simple assay with a natural hot-start reaction. This created pool of extension products of DNA sequence forms amplicons amplified by
80:
PEA is derived from the proximity ligation assay (PLA) which uses a DNA ligase enzyme to unite the sequences of the proximity probes instead of a DNA polymerase. PLA is also suitable for multiplexing, but suffers from enzymatic sample variable inhibition of ligase enzymes from components of serum and
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by DNA-tag counting. PEA enables the detection of many proteins simultaneously (so called multiplexing) due to the readout requiring the combination of two correctly bound antibodies per protein to generate a detectable DNA sequence from the extension reaction. Only cognate pairs of sequence are
47:. Biomarkers and biomarker signature combinations, are useful for determining disease states and drug efficacy. Most methods for detecting proteins involve the use of a solid phase for first capturing and immobilizing the protein analyte, where in one or a few proteins are quantified, such as
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Lundberg, M.; Thorsen, S. B.; Assarsson, E.; Villablanca, A.; Tran, B.; Gee, N.; Knowles, M.; Nielsen, B. S.; González Couto, E.; Martin, R.; Nilsson, O.; Fermer, C.; Schlingemann, J.; Christensen, I. J.; Nielsen, H. J.; Ekström, B.; Andersson, C.; Gustafsson, M.; Brunner, N.; Stenvang, J.;
51:. In contrast, PEA is performed without a solid phase in a homogeneous one tube reaction solution where in sets of antibodies coupled to unique DNA sequence tags, so called proximity probes, work in pairs specific for each target protein. PEA is often performed using
72:
detected as true signal, enabling multiplexing beyond solid phase capture methods limited at around 30 proteins at a time. The DNA amplification power also enable minute sample volumes even below one microliter. PEA has been used in over 1000 research publications.
461:
Assarsson, E.; Lundberg, M.; Holmquist, G.; Björkesten, J.; Thorsen, S. B.; Ekman, D.; Eriksson, A.; Rennel
Dickens, E.; Ohlsson, S.; Edfeldt, G.; Andersson, A. C.; Lindstedt, P.; Stenvang, J.; Gullberg, M.; Fredriksson, S. (2014).
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Wik, Lotta; Nordberg, Niklas; Broberg, John; Björkesten, Johan; Assarsson, Erika; Henriksson, Sara; Grundberg, Ida; Pettersson, Erik; Westerberg, Christina; Liljeroth, Elin; Falck, Adam; Lundberg, Martin (2021).
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plasma samples. DNA polymerase enzymes do not suffer the same inhibition and is also readily multiplexable and has been multiplexed up to 384 proteins. PEA performance is temperature sensitive as it is a
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Fredriksson, S.; Gullberg, M.; Jarvius, J.; Olsson, C.; Pietras, K.; Gústafsdóttir, S. M.; Ostman, A.; Landegren, U. (2002). "Protein detection using proximity-dependent DNA ligation assays".
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where each amplicon sequence corresponds to a target proteins identity and the amount reflects its quantity. Subsequently, these amplicons are detected and quantified by either
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Fredriksson, S.; Dixon, W.; Ji, H.; Koong, A. C.; Mindrinos, M.; Davis, R. W. (2007). "Multiplexed protein detection by proximity ligation for cancer biomarker validation".
43:, specifically affinity proteomics, where in one searches for differences in the abundance of many specific proteins in blood for use as a
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Gullberg, M.; Gústafsdóttir, S. M.; Schallmeiner, E.; Jarvius, J.; Bjarnegård, M.; Betsholtz, C.; Landegren, U.; Fredriksson, S. (2004).
85:-based reaction. So the use of hyper-thermostable polymerases with no activity at room temperature supports bench top reaction assembly.
104:"Homogeneous antibody-based proximity extension assays provide sensitive and specific detection of low-abundant proteins in human blood"
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409:"Multiplexed homogeneous proximity ligation assays for high-throughput protein biomarker research in serological material"
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524:"Proximity Extension Assay in Combination with Next-Generation Sequencing for High-throughput Proteome-wide Analysis"
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153:"Emerging Affinity-Based Proteomic Technologies for Large-Scale Plasma Profiling in Cardiovascular Disease"
464:"Homogenous 96-plex PEA immunoassay exhibiting high sensitivity, specificity, and excellent scalability"
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277:"Olink announces milestone achievement of 1,000 peer-reviewed articles citing use of PEA technology"
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Lundberg, M.; Eriksson, A.; Tran, B.; Assarsson, E.; Fredriksson, S. (2011).
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349:"Cytokine detection by antibody-based proximity ligation"
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151:Smith, J. Gustav; Gerszten, Robert E. (2017).
39:. The method is used in the research field of
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31:present in a biological sample such as
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528:Molecular & Cellular Proteomics
413:Molecular & Cellular Proteomics
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291:"1,000 peer-reviewed publication"
279:(Press release). 3 November 2022.
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21:proximity extension assay
407:Fredriksson, S. (2011).
16:Protein detection method
426:10.1074/mcp.M110.004978
374:10.1073/pnas.0400552101
108:Nucleic Acids Research
306:Nature Biotechnology
480:2014PLoSO...995192A
365:2004PNAS..101.8420G
318:10.1038/nbt0502-473
419:(4): M110.004978.
293:. 3 November 2022.
120:10.1093/nar/gkr424
359:(22): 8420–8424.
236:10.1038/nmeth1020
163:(17): 1651–1664.
83:DNA hybridization
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157:Circulation
76:Development
57:immunoassay
578:Proteomics
572:Categories
534:: 100168.
89:References
53:antibodies
41:proteomics
45:biomarker
558:34715355
508:24755770
468:PLOS ONE
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187:28438806
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29:proteins
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