Validation of an enzyme-linked immunosorbent assay for the quantification of citrullinated histone H3 as a marker for neutrophil extracellular traps in human plasma
العنوان: | 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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المؤلفون: | Thålin, Charlotte, Daleskog, Maud, Paues Göransson, Sophie, Schatzberg, Daphne, Lasselin, Julie, Laska, Ann-Charlotte, Kallner, Anders, Helleday, Thomas, Wallén, Håkan, Demers, Mélanie |
المصدر: | Immunologic research. 65(3):706-712 |
مصطلحات موضوعية: | Elisa, H3Cit, Human plasma, LPS-induced inflammation, NETs, PAD4 |
الوصف: | 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. |
وصف الملف: | |
URL الوصول: | https://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-141474 https://doi.org/10.1007/s12026-017-8905-3 |
قاعدة البيانات: | SwePub |
تدمد: | 0257277X 15590755 |
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DOI: | 10.1007/s12026-017-8905-3 |