Methodological aspects of ELISA analysis of thioredoxin 1 in human plasma and cerebrospinal fluid
Thioredoxin-1 (Trx1) is a protein antioxidant involved in major cellular processes. Increased plasma levels of Trx1 have been associated with human diseases suggesting that Trx1 is a marker for oxidative stress with putative clinical use. However, the reported mean levels of Trx1 in the control coho...
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| description | Thioredoxin-1 (Trx1) is a protein antioxidant involved in major cellular processes. Increased plasma levels of Trx1 have been associated with human diseases suggesting that Trx1 is a marker for oxidative stress with putative clinical use. However, the reported mean levels of Trx1 in the control cohorts vary a hundred-fold between studies (0.8-87 ng/ml), possibly due to methodological differences between the capture ELISA used in the different studies. The aim of this study was to investigate methodological aspects related to the ELISA measurement of Trx1. ELISAs utilizing different capture and detection combinations of antibodies to Trx1 and as well as recombinant human (rh) Trx1 standards from two sources were characterized. The different ELISAs were subsequently used to measure Trx1 in human plasma and cerebrospinal fluid samples (CSF) from healthy donors and from patients with various neurological diagnoses. The Trx1 standards differed in their content of monomeric and oligomeric Trx1, which affected the ELISAs composed of different antibody combinations. Thus, the levels of Trx1 determined in human plasma and CSF samples varied depending on the antibody used in the ELISAs and on the rhTrx1 standard. Furthermore, the relevance of preventing interference by heterophilic antibodies (HA) in human plasma and CSF was investigated. The addition of a HA blocking buffer to human samples drastically reduced the ELISA signals in many samples showing that HA are likely to cause false positive results unless they are blocked. In conclusion, the study shows that the design of a Trx1 ELISA in regards to antibodies and standards used has an impact on the measured Trx1 levels. Importantly, analyses of human plasma and CSF without preventing HA interference may obscure the obtained data. Overall, the results of this study are crucial for the improvement of future studies on the association of Trx1 levels with various diseases. |
| doi_str_mv | 10.1371/journal.pone.0103554 |
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Increased plasma levels of Trx1 have been associated with human diseases suggesting that Trx1 is a marker for oxidative stress with putative clinical use. However, the reported mean levels of Trx1 in the control cohorts vary a hundred-fold between studies (0.8-87 ng/ml), possibly due to methodological differences between the capture ELISA used in the different studies. The aim of this study was to investigate methodological aspects related to the ELISA measurement of Trx1. ELISAs utilizing different capture and detection combinations of antibodies to Trx1 and as well as recombinant human (rh) Trx1 standards from two sources were characterized. The different ELISAs were subsequently used to measure Trx1 in human plasma and cerebrospinal fluid samples (CSF) from healthy donors and from patients with various neurological diagnoses. The Trx1 standards differed in their content of monomeric and oligomeric Trx1, which affected the ELISAs composed of different antibody combinations. Thus, the levels of Trx1 determined in human plasma and CSF samples varied depending on the antibody used in the ELISAs and on the rhTrx1 standard. Furthermore, the relevance of preventing interference by heterophilic antibodies (HA) in human plasma and CSF was investigated. The addition of a HA blocking buffer to human samples drastically reduced the ELISA signals in many samples showing that HA are likely to cause false positive results unless they are blocked. In conclusion, the study shows that the design of a Trx1 ELISA in regards to antibodies and standards used has an impact on the measured Trx1 levels. Importantly, analyses of human plasma and CSF without preventing HA interference may obscure the obtained data. Overall, the results of this study are crucial for the improvement of future studies on the association of Trx1 levels with various diseases.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0103554</identifier><identifier>PMID: 25075746</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Adult ; Analysis ; Antibodies ; Antibodies - immunology ; Antioxidants ; Biological activity ; Biology and Life Sciences ; Blood plasma ; Blotting, Western ; Cerebrospinal fluid ; Disease ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Immunoassay ; Immunoglobulins ; Interference ; Kinases ; Laboratories ; Male ; Medicin och hälsovetenskap ; Medicine and Health Sciences ; Middle Aged ; Nervous System Diseases - cerebrospinal fluid ; Nervous System Diseases - diagnosis ; Oxidative Stress ; Plasma levels ; Proteins ; Psychosis ; Recombinant Proteins - biosynthesis ; Recombinant Proteins - genetics ; Recombinant Proteins - immunology ; Schizophrenia ; Thioredoxin ; Thioredoxins ; Thioredoxins - analysis ; Thioredoxins - blood ; Thioredoxins - cerebrospinal fluid</subject><ispartof>PloS one, 2014-07, Vol.9 (7), p.e103554-e103554</ispartof><rights>COPYRIGHT 2014 Public Library of Science</rights><rights>2014 Lundberg et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 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Increased plasma levels of Trx1 have been associated with human diseases suggesting that Trx1 is a marker for oxidative stress with putative clinical use. However, the reported mean levels of Trx1 in the control cohorts vary a hundred-fold between studies (0.8-87 ng/ml), possibly due to methodological differences between the capture ELISA used in the different studies. The aim of this study was to investigate methodological aspects related to the ELISA measurement of Trx1. ELISAs utilizing different capture and detection combinations of antibodies to Trx1 and as well as recombinant human (rh) Trx1 standards from two sources were characterized. The different ELISAs were subsequently used to measure Trx1 in human plasma and cerebrospinal fluid samples (CSF) from healthy donors and from patients with various neurological diagnoses. The Trx1 standards differed in their content of monomeric and oligomeric Trx1, which affected the ELISAs composed of different antibody combinations. Thus, the levels of Trx1 determined in human plasma and CSF samples varied depending on the antibody used in the ELISAs and on the rhTrx1 standard. Furthermore, the relevance of preventing interference by heterophilic antibodies (HA) in human plasma and CSF was investigated. The addition of a HA blocking buffer to human samples drastically reduced the ELISA signals in many samples showing that HA are likely to cause false positive results unless they are blocked. In conclusion, the study shows that the design of a Trx1 ELISA in regards to antibodies and standards used has an impact on the measured Trx1 levels. Importantly, analyses of human plasma and CSF without preventing HA interference may obscure the obtained data. 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Increased plasma levels of Trx1 have been associated with human diseases suggesting that Trx1 is a marker for oxidative stress with putative clinical use. However, the reported mean levels of Trx1 in the control cohorts vary a hundred-fold between studies (0.8-87 ng/ml), possibly due to methodological differences between the capture ELISA used in the different studies. The aim of this study was to investigate methodological aspects related to the ELISA measurement of Trx1. ELISAs utilizing different capture and detection combinations of antibodies to Trx1 and as well as recombinant human (rh) Trx1 standards from two sources were characterized. The different ELISAs were subsequently used to measure Trx1 in human plasma and cerebrospinal fluid samples (CSF) from healthy donors and from patients with various neurological diagnoses. The Trx1 standards differed in their content of monomeric and oligomeric Trx1, which affected the ELISAs composed of different antibody combinations. Thus, the levels of Trx1 determined in human plasma and CSF samples varied depending on the antibody used in the ELISAs and on the rhTrx1 standard. Furthermore, the relevance of preventing interference by heterophilic antibodies (HA) in human plasma and CSF was investigated. The addition of a HA blocking buffer to human samples drastically reduced the ELISA signals in many samples showing that HA are likely to cause false positive results unless they are blocked. In conclusion, the study shows that the design of a Trx1 ELISA in regards to antibodies and standards used has an impact on the measured Trx1 levels. Importantly, analyses of human plasma and CSF without preventing HA interference may obscure the obtained data. Overall, the results of this study are crucial for the improvement of future studies on the association of Trx1 levels with various diseases.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>25075746</pmid><doi>10.1371/journal.pone.0103554</doi><oa>free_for_read</oa></addata></record> |
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| recordid | cdi_plos_journals_1549924628 |
| source | Public Library of Science (PLoS) Journals Open Access; PubMed Central (Open access); Elektronische Zeitschriftenbibliothek (Open access); MEDLINE; Full-Text Journals in Chemistry (Open access); Directory of Open Access Journals; SWEPUB Freely available online |
| subjects | Adult Analysis Antibodies Antibodies - immunology Antioxidants Biological activity Biology and Life Sciences Blood plasma Blotting, Western Cerebrospinal fluid Disease Enzyme-Linked Immunosorbent Assay Female Humans Immunoassay Immunoglobulins Interference Kinases Laboratories Male Medicin och hälsovetenskap Medicine and Health Sciences Middle Aged Nervous System Diseases - cerebrospinal fluid Nervous System Diseases - diagnosis Oxidative Stress Plasma levels Proteins Psychosis Recombinant Proteins - biosynthesis Recombinant Proteins - genetics Recombinant Proteins - immunology Schizophrenia Thioredoxin Thioredoxins Thioredoxins - analysis Thioredoxins - blood Thioredoxins - cerebrospinal fluid |
| title | Methodological aspects of ELISA analysis of thioredoxin 1 in human plasma and cerebrospinal fluid |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-01-03T17%3A39%3A53IST&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=Methodological%20aspects%20of%20ELISA%20analysis%20of%20thioredoxin%201%20in%20human%20plasma%20and%20cerebrospinal%20fluid&rft.jtitle=PloS%20one&rft.au=Lundberg,%20Mathias&rft.date=2014-07-30&rft.volume=9&rft.issue=7&rft.spage=e103554&rft.epage=e103554&rft.pages=e103554-e103554&rft.issn=1932-6203&rft.eissn=1932-6203&rft_id=info:doi/10.1371/journal.pone.0103554&rft_dat=%3Cgale_plos_%3EA417148437%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=1549924628&rft_id=info:pmid/25075746&rft_galeid=A417148437&rft_doaj_id=oai_doaj_org_article_b3ce300e2ed148a7a00c3ee0c655eba0&rfr_iscdi=true |