A high-sensitivity electrochemiluminescence-based ELISA for the measurement of the oxidative stress biomarker, 3-nitrotyrosine, in human blood serum and cells


The generation of 3-nitrotyrosine, within proteins, is a post-translational modification resulting from oxidative or nitrative stress. It has been suggested that this modification could be used as a biomarker for inflammatory diseases. Despite the superiority of mass spectrometry-based determinations of nitrotyrosine, in a high-throughput clinical setting the measurement of nitrotyrosine by an enzyme-linked immunosorbent assay (ELISA) is likely to be more cost-effective. ELISAs offer an alternative means to detect nitrotyrosine, but many commercially available ELISAs are insufficiently sensitive to detect nitrotyrosine in healthy human serum. Here, we report the development, validation and clinical application of a novel electrochemiluminescence-based ELISA for nitrotyrosine which provides superior sensitivity (e.g. a 50-fold increase in sensitivity compared with one of the tested commercial colorimetric ELISAs). This nitrotyrosine ELISA has the following characteristics: a lower limit of quantitation of 0.04 nM nitrated albumin equivalents; intra- and inter-assay coefficients of variation of 6.5% and 11.3%, respectively; a mean recovery of 106 ± 3% and a mean linearity of 0.998 ± 0.001. Far higher nitration levels were measured in normal human blood cell populations when compared to plasma. Mass spectrometry was used to validate the new ELISA method. The analysis of the same set of chemically modified albumin samples using the ELISA method and mass spectrometry showed good agreement for the relative levels of nitration present in each sample. The assay was applied to serum samples from patients undergoing elective surgery which induces the human inflammatory response. Matched samples were collected before and one day after surgery. An increase in nitration was detected following surgery (median (IQR): 0.59 (0.00–1.34) and 0.97 (0.00–1.70) nitrotyrosine (fmol of nitrated albumin equivalents/mg protein) for pre- and post-surgery respectively. The reported assay is suitable for nitrotyrosine determination in patient serum samples, and may also be applicable as a means to determine oxidative stress in primary and cultured cell populations.

Publication DOI: https://doi.org/10.1016/j.freeradbiomed.2018.03.026
Divisions: College of Health & Life Sciences > Aston Pharmacy School
College of Health & Life Sciences
College of Health & Life Sciences > School of Biosciences > Cellular and Molecular Biomedicine
College of Health & Life Sciences > School of Biosciences
College of Health & Life Sciences > Chronic and Communicable Conditions
Additional Information: © 2018, Elsevier. Licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International http://creativecommons.org/licenses/by-nc-nd/4.0/
Uncontrolled Keywords: 3-nitrotyrosine,oxidative stress,nitrative stress,peroxynitrite, inflammation, human serum,enzyme-linked immunosorbent assay,mass spectrometry
Publication ISSN: 1873-4596
Last Modified: 18 Jul 2024 07:06
Date Deposited: 19 Mar 2018 08:35
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Related URLs: https://www.sci ... 1291?via%3Dihub (Publisher URL)
PURE Output Type: Article
Published Date: 2018-05-20
Published Online Date: 2018-03-17
Accepted Date: 2018-03-14
Authors: Knight, Annie R.
Taylor, Emma
Lukaszewski, Roman
Tveen Jensen, Karina
Jones, Helen E.
Carré, Jane E.
supov, Michail N. I
Littlechild, Jennifer A.
Bailey, Stephen J.
Brewer, Emily
McDonald, Timothy J.
Pitt, Andrew R (ORCID Profile 0000-0003-3619-6503)
Spickett, Corinne M (ORCID Profile 0000-0003-4054-9279)
Winyard, Paul G

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