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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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Autores principales: Lundberg, Mathias, Curbo, Sophie, Reiser, Kathrin, Masterman, Thomas, Braesch-Andersen, Sten, Areström, Irene, Ahlborg, Niklas
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4116216/
https://www.ncbi.nlm.nih.gov/pubmed/25075746
http://dx.doi.org/10.1371/journal.pone.0103554
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author Lundberg, Mathias
Curbo, Sophie
Reiser, Kathrin
Masterman, Thomas
Braesch-Andersen, Sten
Areström, Irene
Ahlborg, Niklas
author_facet Lundberg, Mathias
Curbo, Sophie
Reiser, Kathrin
Masterman, Thomas
Braesch-Andersen, Sten
Areström, Irene
Ahlborg, Niklas
author_sort Lundberg, Mathias
collection PubMed
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.
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spelling pubmed-41162162014-08-04 Methodological Aspects of ELISA Analysis of Thioredoxin 1 in Human Plasma and Cerebrospinal Fluid Lundberg, Mathias Curbo, Sophie Reiser, Kathrin Masterman, Thomas Braesch-Andersen, Sten Areström, Irene Ahlborg, Niklas PLoS One Research Article 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. Public Library of Science 2014-07-30 /pmc/articles/PMC4116216/ /pubmed/25075746 http://dx.doi.org/10.1371/journal.pone.0103554 Text en © 2014 Lundberg et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Lundberg, Mathias
Curbo, Sophie
Reiser, Kathrin
Masterman, Thomas
Braesch-Andersen, Sten
Areström, Irene
Ahlborg, Niklas
Methodological Aspects of ELISA Analysis of Thioredoxin 1 in Human Plasma and Cerebrospinal Fluid
title Methodological Aspects of ELISA Analysis of Thioredoxin 1 in Human Plasma and Cerebrospinal Fluid
title_full Methodological Aspects of ELISA Analysis of Thioredoxin 1 in Human Plasma and Cerebrospinal Fluid
title_fullStr Methodological Aspects of ELISA Analysis of Thioredoxin 1 in Human Plasma and Cerebrospinal Fluid
title_full_unstemmed Methodological Aspects of ELISA Analysis of Thioredoxin 1 in Human Plasma and Cerebrospinal Fluid
title_short Methodological Aspects of ELISA Analysis of Thioredoxin 1 in Human Plasma and Cerebrospinal Fluid
title_sort methodological aspects of elisa analysis of thioredoxin 1 in human plasma and cerebrospinal fluid
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4116216/
https://www.ncbi.nlm.nih.gov/pubmed/25075746
http://dx.doi.org/10.1371/journal.pone.0103554
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