Homogenous 96-plex PEA immunoassay exhibiting high sensitivity, specificity, and excellent scalability
Medical research is developing an ever greater need for comprehensive high-quality data generation to realize the promises of personalized health care based on molecular biomarkers. The nucleic acid proximity-based methods proximity ligation and proximity extension assays have, with their dual repor...
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| Veröffentlicht in: | PloS one 2014-04, Vol.9 (4), p.e95192-e95192 |
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| creator | Assarsson, Erika Lundberg, Martin Holmquist, Göran Björkesten, Johan Thorsen, Stine Bucht Ekman, Daniel Eriksson, Anna Rennel Dickens, Emma Ohlsson, Sandra Edfeldt, Gabriella Andersson, Ann-Catrin Lindstedt, Patrik Stenvang, Jan Gullberg, Mats Fredriksson, Simon |
| description | Medical research is developing an ever greater need for comprehensive high-quality data generation to realize the promises of personalized health care based on molecular biomarkers. The nucleic acid proximity-based methods proximity ligation and proximity extension assays have, with their dual reporters, shown potential to relieve the shortcomings of antibodies and their inherent cross-reactivity in multiplex protein quantification applications. The aim of the present study was to develop a robust 96-plex immunoassay based on the proximity extension assay (PEA) for improved high throughput detection of protein biomarkers. This was enabled by: (1) a modified design leading to a reduced number of pipetting steps compared to the existing PEA protocol, as well as improved intra-assay precision; (2) a new enzymatic system that uses a hyper-thermostabile enzyme, Pwo, for uniting the two probes allowing for room temperature addition of all reagents and improved the sensitivity; (3) introduction of an inter-plate control and a new normalization procedure leading to improved inter-assay precision (reproducibility). The multiplex proximity extension assay was found to perform well in complex samples, such as serum and plasma, and also in xenografted mice and resuspended dried blood spots, consuming only 1 µL sample per test. All-in-all, the development of the current multiplex technique is a step toward robust high throughput protein marker discovery and research. |
| doi_str_mv | 10.1371/journal.pone.0095192 |
| format | Article |
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The nucleic acid proximity-based methods proximity ligation and proximity extension assays have, with their dual reporters, shown potential to relieve the shortcomings of antibodies and their inherent cross-reactivity in multiplex protein quantification applications. The aim of the present study was to develop a robust 96-plex immunoassay based on the proximity extension assay (PEA) for improved high throughput detection of protein biomarkers. This was enabled by: (1) a modified design leading to a reduced number of pipetting steps compared to the existing PEA protocol, as well as improved intra-assay precision; (2) a new enzymatic system that uses a hyper-thermostabile enzyme, Pwo, for uniting the two probes allowing for room temperature addition of all reagents and improved the sensitivity; (3) introduction of an inter-plate control and a new normalization procedure leading to improved inter-assay precision (reproducibility). The multiplex proximity extension assay was found to perform well in complex samples, such as serum and plasma, and also in xenografted mice and resuspended dried blood spots, consuming only 1 µL sample per test. All-in-all, the development of the current multiplex technique is a step toward robust high throughput protein marker discovery and research.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0095192</identifier><identifier>PMID: 24755770</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Analysis ; Animals ; Antibodies ; Antigens ; Binding sites ; Biology ; Biology and Life Sciences ; Biomarkers ; Blood Proteins - metabolism ; Breast cancer ; Cloning ; Cross Reactions ; Cross-reactivity ; Deoxyribonucleic acid ; Design modifications ; DNA ; DNA polymerase ; DNA-Directed DNA Polymerase - metabolism ; Dried Blood Spot Testing ; Enzyme Stability ; Female ; Health care ; Heterografts ; Humans ; Immunoassay ; Immunoassay - methods ; Immunoglobulins ; Medical research ; Medicine and Health Sciences ; Mice, Nude ; Multiplexing ; Oligonucleotides - metabolism ; Polymerase Chain Reaction - methods ; Protein research ; Proteins ; Proteomics ; Proximity ; Reagents ; Reference services ; Reproducibility ; Research and Analysis Methods ; Sensitivity ; Sensitivity and Specificity ; Spots ; Temperature ; Xenografts</subject><ispartof>PloS one, 2014-04, Vol.9 (4), p.e95192-e95192</ispartof><rights>COPYRIGHT 2014 Public Library of Science</rights><rights>2014 Assarsson et al. 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The multiplex proximity extension assay was found to perform well in complex samples, such as serum and plasma, and also in xenografted mice and resuspended dried blood spots, consuming only 1 µL sample per test. All-in-all, the development of the current multiplex technique is a step toward robust high throughput protein marker discovery and research.</description><subject>Analysis</subject><subject>Animals</subject><subject>Antibodies</subject><subject>Antigens</subject><subject>Binding sites</subject><subject>Biology</subject><subject>Biology and Life Sciences</subject><subject>Biomarkers</subject><subject>Blood Proteins - metabolism</subject><subject>Breast cancer</subject><subject>Cloning</subject><subject>Cross Reactions</subject><subject>Cross-reactivity</subject><subject>Deoxyribonucleic acid</subject><subject>Design modifications</subject><subject>DNA</subject><subject>DNA polymerase</subject><subject>DNA-Directed DNA Polymerase - metabolism</subject><subject>Dried Blood Spot Testing</subject><subject>Enzyme Stability</subject><subject>Female</subject><subject>Health care</subject><subject>Heterografts</subject><subject>Humans</subject><subject>Immunoassay</subject><subject>Immunoassay 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96-plex PEA immunoassay exhibiting high sensitivity, specificity, and excellent scalability</title><author>Assarsson, Erika ; Lundberg, Martin ; Holmquist, Göran ; Björkesten, Johan ; Thorsen, Stine Bucht ; Ekman, Daniel ; Eriksson, Anna ; Rennel Dickens, Emma ; Ohlsson, Sandra ; Edfeldt, Gabriella ; Andersson, Ann-Catrin ; Lindstedt, Patrik ; Stenvang, Jan ; Gullberg, Mats ; Fredriksson, Simon</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c743t-8710640492ec3e4d2b179dac2d1d8b7ef3b5b879f5b32f1d89f4b71da1650863</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2014</creationdate><topic>Analysis</topic><topic>Animals</topic><topic>Antibodies</topic><topic>Antigens</topic><topic>Binding sites</topic><topic>Biology</topic><topic>Biology and Life Sciences</topic><topic>Biomarkers</topic><topic>Blood Proteins - metabolism</topic><topic>Breast cancer</topic><topic>Cloning</topic><topic>Cross 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The nucleic acid proximity-based methods proximity ligation and proximity extension assays have, with their dual reporters, shown potential to relieve the shortcomings of antibodies and their inherent cross-reactivity in multiplex protein quantification applications. The aim of the present study was to develop a robust 96-plex immunoassay based on the proximity extension assay (PEA) for improved high throughput detection of protein biomarkers. This was enabled by: (1) a modified design leading to a reduced number of pipetting steps compared to the existing PEA protocol, as well as improved intra-assay precision; (2) a new enzymatic system that uses a hyper-thermostabile enzyme, Pwo, for uniting the two probes allowing for room temperature addition of all reagents and improved the sensitivity; (3) introduction of an inter-plate control and a new normalization procedure leading to improved inter-assay precision (reproducibility). The multiplex proximity extension assay was found to perform well in complex samples, such as serum and plasma, and also in xenografted mice and resuspended dried blood spots, consuming only 1 µL sample per test. All-in-all, the development of the current multiplex technique is a step toward robust high throughput protein marker discovery and research.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>24755770</pmid><doi>10.1371/journal.pone.0095192</doi><oa>free_for_read</oa></addata></record> |
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| identifier | ISSN: 1932-6203 |
| ispartof | PloS one, 2014-04, Vol.9 (4), p.e95192-e95192 |
| issn | 1932-6203 1932-6203 |
| language | eng |
| recordid | cdi_plos_journals_1518469593 |
| source | MEDLINE; DOAJ Directory of Open Access Journals; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; PubMed Central; Free Full-Text Journals in Chemistry; Public Library of Science (PLoS) |
| subjects | Analysis Animals Antibodies Antigens Binding sites Biology Biology and Life Sciences Biomarkers Blood Proteins - metabolism Breast cancer Cloning Cross Reactions Cross-reactivity Deoxyribonucleic acid Design modifications DNA DNA polymerase DNA-Directed DNA Polymerase - metabolism Dried Blood Spot Testing Enzyme Stability Female Health care Heterografts Humans Immunoassay Immunoassay - methods Immunoglobulins Medical research Medicine and Health Sciences Mice, Nude Multiplexing Oligonucleotides - metabolism Polymerase Chain Reaction - methods Protein research Proteins Proteomics Proximity Reagents Reference services Reproducibility Research and Analysis Methods Sensitivity Sensitivity and Specificity Spots Temperature Xenografts |
| title | Homogenous 96-plex PEA immunoassay exhibiting high sensitivity, specificity, and excellent scalability |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-02-03T15%3A40%3A30IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-gale_plos_&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Homogenous%2096-plex%20PEA%20immunoassay%20exhibiting%20high%20sensitivity,%20specificity,%20and%20excellent%20scalability&rft.jtitle=PloS%20one&rft.au=Assarsson,%20Erika&rft.date=2014-04-01&rft.volume=9&rft.issue=4&rft.spage=e95192&rft.epage=e95192&rft.pages=e95192-e95192&rft.issn=1932-6203&rft.eissn=1932-6203&rft_id=info:doi/10.1371/journal.pone.0095192&rft_dat=%3Cgale_plos_%3EA375584526%3C/gale_plos_%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=1518469593&rft_id=info:pmid/24755770&rft_galeid=A375584526&rft_doaj_id=oai_doaj_org_article_e2de90278afd47aebacc53f5871053c0&rfr_iscdi=true |