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Validation of an en...
Validation of an enzyme-linked immunosorbent assay for the quantification of citrullinated histone H3 as a marker for neutrophil extracellular traps in human plasma
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- Thålin, Charlotte (author)
- Karolinska Institutet
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Daleskog, Maud (author)
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Paues Göransson, Sophie (author)
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Schatzberg, Daphne (author)
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- Lasselin, Julie (author)
- Stockholms universitet,Stressforskningsinstitutet,Karolinska Institutet, Sweden
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- Laska, Ann-Charlotte (author)
- Karolinska Institutet
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- Kallner, Anders (author)
- Karolinska Institutet
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- Helleday, Thomas (author)
- Karolinska Institutet
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- Wallén, Håkan (author)
- Karolinska Institutet
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Demers, Mélanie (author)
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(creator_code:org_t)
- 2017-02-04
- 2017
- English.
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In: Immunologic research. - : Springer Science and Business Media LLC. - 0257-277X .- 1559-0755. ; 65:3, s. 706-712
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Abstract
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- There is an emerging interest in the diverse functions of neutrophil extracellular traps (NETs) in a variety of disease settings. However, data on circulating NETs rely largely upon surrogate NET markers such as cell-free DNA, nucleosomes, and NET-associated enzymes. Citrullination of histone H3 by peptidyl arginine deiminase 4 (PAD4) is central for NET formation, and citrullinated histone H3 (H3Cit) is considered a NET-specific biomarker. We therefore aimed to optimize and validate a new enzyme-linked immunosorbent assay (ELISA) to quantify the levels of H3Cit in human plasma. A standard curve made of in vitro PAD4-citrullinated histones H3 allows for the quantification of H3Cit in plasma using an anti-histone antibody as capture antibody and an anti-histone H3 citrulline antibody for detection. The assay was evaluated for linearity, stability, specificity, and precision on plasma samples obtained from a human model of inflammation before and after lipopolysaccharide injection. The results revealed linearity and high specificity demonstrated by the inability of detecting non-citrullinated histone H3. Coefficients of variation for intra- and inter-assay variability ranged from 2.1 to 5.1% and from 5.8 to 13.5%, respectively, allowing for a high precision. Furthermore, our results support an inflammatory induction of a systemic NET burden by showing, for the first time, clear intra-individual elevations of plasma H3Cit in a human model of lipopolysaccharide-induced inflammation. Taken together, our work demonstrates the development of a new method for the quantification of H3Cit by ELISA that can reliably be used for the detection of NETs in human plasma.
Subject headings
- MEDICIN OCH HÄLSOVETENSKAP -- Medicinska och farmaceutiska grundvetenskaper -- Immunologi inom det medicinska området (hsv//swe)
- MEDICAL AND HEALTH SCIENCES -- Basic Medicine -- Immunology in the medical area (hsv//eng)
- MEDICIN OCH HÄLSOVETENSKAP -- Klinisk medicin -- Reumatologi och inflammation (hsv//swe)
- MEDICAL AND HEALTH SCIENCES -- Clinical Medicine -- Rheumatology and Autoimmunity (hsv//eng)
Keyword
- Elisa
- H3Cit
- Human plasma
- LPS-induced inflammation
- NETs
- PAD4
Publication and Content Type
- ref (subject category)
- art (subject category)
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- By the author/editor
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Thålin, Charlott ...
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Daleskog, Maud
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Paues Göransson, ...
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Schatzberg, Daph ...
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Lasselin, Julie
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Laska, Ann-Charl ...
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show more...
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Kallner, Anders
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Helleday, Thomas
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Wallén, Håkan
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Demers, Mélanie
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show less...
- About the subject
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- MEDICAL AND HEALTH SCIENCES
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MEDICAL AND HEAL ...
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and Basic Medicine
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and Immunology in th ...
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- MEDICAL AND HEALTH SCIENCES
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MEDICAL AND HEAL ...
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and Clinical Medicin ...
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and Rheumatology and ...
- Articles in the publication
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Immunologic rese ...
- By the university
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Stockholm University
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Karolinska Institutet