Methodological aspects of ELISA analysis of thioredoxin 1 in human plasma and cerebrospinal fluid.

Mathias Lundberg,Thomas Masterman,Sten Braesch-Andersen,Kathrin Reiser,Sophie Curbo,Irene Areström,Niklas Ahlborg,Markus Reindl

doi:10.1371/journal.pone.0103554

Mathias Lundberg, Thomas Masterman + Show 6 more

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https://doi.org/10.1371/journal.pone.0103554

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Abstract

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.

Highlights

Thioredoxin-1 (Trx1) is a 12 kDa ubiquitous oxido-reductase protein and is despite its small size a biochemically complex protein that catalyses protein disulfide reductions [1]
Antibodies and recombinant proteins Antibodies used for the ELISA as well as for Western blotting were the Protein G-purified mouse monoclonal antibody 2G11 (Mabtech, Nacka Strand, Sweden), [23] and a goat polyclonal antibody to human Trx1 purified on human Trx1 (IMCO, Stockholm, Sweden)
When analysing the DTT-treated Trx1 in ELISA, the concentration of DTT was below 50 nM at the highest concentration of Trx1 tested. 5 mM ethylenediaminetetraacetic acid (EDTA) was added to all ELISA buffers when analysing DTTtreated Trx1; the inclusion of EDTA in the buffers did not have any impact on the detection of Trx1 not treated with DTT

Summary

Introduction

Thioredoxin-1 (Trx1) is a 12 kDa ubiquitous oxido-reductase protein and is despite its small size a biochemically complex protein that catalyses protein disulfide reductions [1]. Trx has three additional structural cysteine residues outside its active site that may form intra- and/or intermolecular disulfide bonds. Trx plays a major role in keeping the intracellular milieu in a reduced state [3] and has a specific activity in regulating the bioactivity of numerous proteins including apoptosis factors, transcription factors and receptors [5]. It is involved in the defence against oxidative stress and its expression is strongly induced in response to it [6,7]

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