Validation of an enzyme-linked immunosorbent assay for the quantification of citrullinated histone H3 as a marker for neutrophil extracellular traps in human plasma
| Autor: | Charlotte Thålin, Håkan Wallén, Sophie Paues Göransson, Daphne Schatzberg, Thomas Helleday, Ann Charlotte Laska, Julie Lasselin, Melanie Demers, Anders Kallner, Maud Daleskog |
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| Rok vydání: | 2017 |
| Předmět: |
Lipopolysaccharides
0301 basic medicine Neutrophils H3Cit Immunology Enzyme-Linked Immunosorbent Assay Elisa LPS-induced inflammation Extracellular Traps Sensitivity and Specificity Histones Plasma 03 medical and health sciences Histone H3 chemistry.chemical_compound Protein-Arginine Deiminase Type 4 Citrulline Humans Nucleosome PAD4 Inflammation Observer Variation biology Citrullination NETs Neutrophil extracellular traps Molecular biology 030104 developmental biology Histone Biochemistry chemistry Human plasma Protein-Arginine Deiminases biology.protein Feasibility Studies Biomarker (medicine) Original Article Antibody Biomarkers |
| Zdroj: | Immunologic Research |
| ISSN: | 1559-0755 0257-277X |
| DOI: | 10.1007/s12026-017-8905-3 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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