Rapid and sensitive determination of protein-nitrotyrosine by ELISA: Application to human plasma
Abstract
3-Nitrotyrosine (3NT) is known as an important indicator of nitrosative stress and has been linked to various diseases. Our aim was to develop an indirect ELISA (enzyme-linked immunosorbent assay) method suitable for the detection of protein-bound 3NT in clinical plasma and serum samples. Nitrated protein standards and reduced protein standards were prepared. Limit of detection was determined for standards; recovery and reproducibility were determined for human plasma samples. The limit of detection for this method is 1.82±0.56 pmol/mg protein. Mean recovery of standards was 95%. 3NT concentration in plasma samples of obese and normal weight subjects was determined to be between 2 pmol/mg and 19 pmol/mg. No time-consuming sample preparation or expensive laboratory equipment is required, and applied antibodies are commercially available. Sensitivity, rapid analysis time, possibilities of high throughput applications and small sample volumes make this ELISA attractive for use in clinical laboratories.
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Weber et al. provided a simple and fast indirect ELISA method for measurement of protein-bound 3-NT in human serum and plasma samples.
“Weber et al. provided a simple and fast indirect ELISA method for the measurement of protein-bound 3-NT in human serum and plasma samples.”
From Protein oxidation - Formation mechanisms, detection and relevance as biomarkers in human diseases (2021) · cites this paper as [196]
Several limitations of ELISA for 3-NT have been highlighted in the literature, including low sensitivity, minor specificity of applied antibodies, susceptibility to errors, and missing detection of free 3-NT.
“several limitations have been highlighted in the literature, such as low sensitivity, minor specificity of applied antibodies, the susceptibility to errors, or missing detection of free 3-NT”
From Protein oxidation - Formation mechanisms, detection and relevance as biomarkers in human diseases (2021) · cites this paper as [196]
The Weber et al. ELISA method has high detection limits, small sample volumes, and opportunity for high throughput screening, making it useful for clinical applications.
“High detection limits, small sample volumes, and the opportunity for high throughput-screening makes this method useful for clinical applications”
From Protein oxidation - Formation mechanisms, detection and relevance as biomarkers in human diseases (2021) · cites this paper as [196]
Cites
Beal, M. F. (2002) · Free Radical Biology and Medicine · doi:10.1016/s0891-5849(02)00780-3 · Cited by 763 · see citing works
Aconitase is readily inactivated by peroxynitrite, but not by its precursor, nitric oxide.
Castro, L., Rodriguez, M., & Radi, R. (1994) · Journal of Biological Chemistry · doi:10.1016/s0021-9258(18)43894-x · Cited by 589 · see citing works
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Protein oxidation - Formation mechanisms, detection and relevance as biomarkers in human diseases
Kehm, R., Baldensperger, T., Raupbach, J., & Höhn, A. (2021) · Redox Biology · doi:10.1016/j.redox.2021.101901 · Cited by 348 · see citing works
“High detection limits, small sample volumes, and the opportunity for high throughput-screening makes this method useful for clinical applications [196].”
Method
- Authors
- Weber, D., Kneschke, N., Grimm, S., Bergheim, I., Breusing, N., & Grune, T.
- Affiliations
- Friedrich Schiller University Jena (DE)
- University of Hohenheim (DE)
- Corresponding author
- Grune, Tilman
- Venue
- Free Radical Research
- Publisher
- Informa UK Limited
- Published
- 2012-02-06
- Volume
- 46
- Issue
- 3
- Pages
- 276-285
- ISSN
- 1029-2470 (ISSN-L)
- 1071-5762
- OpenAlex
- W2088530389
- Cited by
- 47
- References
- 2
- Citations by year
- 2025: 3 · 2024: 3 · 2022: 7 · 2021: 1 · 2020: 4 · 2019: 3 · 2018: 3 · 2017: 6 · 2016: 2 · 2015: 4 · 2014: 5 · 2013: 6
- FWCI
- 1.92
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Free Radical Research (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Redox biology and oxidative stress
- Health Sciences › Medicine › Biochemistry › Antioxidant Activity and Oxidative Stress
- Health Sciences › Medicine › Physiology › Nitric Oxide and Endothelin Effects
- Keywords
- nitrotyrosine, nitrosative stress, indirect ELISA, human plasma, serum, obesity, detection limit, clinical laboratory assay
- MeSH terms
- Adolescent, Adult, Aged, Aged, 80 and over, Animals, Blood Proteins, Cattle, Detergents, Enzyme-Linked Immunosorbent Assay, Female, Humans, Middle Aged, Obesity, Oxidation-Reduction, Plasma, Polysorbates, Preservation, Biological, Reference Standards, Sensitivity and Specificity, Serum Albumin, Bovine, Temperature, Time Factors, Tyrosine, Peroxynitrous Acid, Serum, Protein Stability, Young Adult, Limit of Detection
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