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

Annie R. Knight, Emma Taylor, Roman Lukaszewski, Karina Tveen Jensen, Helen E. Jones, Jane E. Carré, Michail N. I supov, Jennifer A. Littlechild, Stephen J. Bailey, Emily Brewer, Timothy J. McDonald, Andrew R Pitt, Corinne M Spickett, Paul G Winyard

Research output: Contribution to journalArticle

Abstract

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.
LanguageEnglish
JournalFree Radical Biology and Medicine
Early online date17 Mar 2018
DOIs
Publication statusE-pub ahead of print - 17 Mar 2018

Fingerprint

Immunosorbents
Oxidative stress
Biomarkers
Assays
Blood Cells
Oxidative Stress
Blood
Enzyme-Linked Immunosorbent Assay
Enzymes
Surgery
Serum
Nitration
Mass spectrometry
Albumins
Mass Spectrometry
Cells
3-nitrotyrosine
Post Translational Protein Processing
Ambulatory Surgical Procedures
Population

Bibliographical note

© 2018, Elsevier. Licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International http://creativecommons.org/licenses/by-nc-nd/4.0/

Keywords

  • 3-nitrotyrosine
  • oxidative stress
  • nitrative stress
  • peroxynitrite
  • inflammation
  • human serum
  • enzyme-linked immunosorbent assay
  • mass spectrometry

Cite this

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 ; Spickett, Corinne M ; Winyard, Paul G. / A high-sensitivity electrochemiluminescence-based ELISA for the measurement of the oxidative stress biomarker, 3-nitrotyrosine, in human blood serum and cells. In: Free Radical Biology and Medicine. 2018.
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A high-sensitivity electrochemiluminescence-based ELISA for the measurement of the oxidative stress biomarker, 3-nitrotyrosine, in human blood serum and cells. / 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; Spickett, Corinne M; Winyard, Paul G.

In: Free Radical Biology and Medicine, 17.03.2018.

Research output: Contribution to journalArticle

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T1 - A high-sensitivity electrochemiluminescence-based ELISA for the measurement of the oxidative stress biomarker, 3-nitrotyrosine, in human blood serum and cells

AU - Knight, Annie R.

AU - Taylor, Emma

AU - Lukaszewski, Roman

AU - Tveen Jensen, Karina

AU - Jones, Helen E.

AU - Carré, Jane E.

AU - supov, Michail N. I

AU - Littlechild, Jennifer A.

AU - Bailey, Stephen J.

AU - Brewer, Emily

AU - McDonald, Timothy J.

AU - Pitt, Andrew R

AU - Spickett, Corinne M

AU - Winyard, Paul G

N1 - © 2018, Elsevier. Licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International http://creativecommons.org/licenses/by-nc-nd/4.0/

PY - 2018/3/17

Y1 - 2018/3/17

N2 - 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.

AB - 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.

KW - 3-nitrotyrosine

KW - oxidative stress

KW - nitrative stress

KW - peroxynitrite

KW - inflammation

KW - human serum

KW - enzyme-linked immunosorbent assay

KW - mass spectrometry

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