Validation of an enzyme-linked immunosorbent assay for the quantification of citrullinated histone H3 as a marker for neutrophil extracellular traps in human plasma.
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.
Department of Clinical Sciences, Danderyd Hospital, Division of Internal Medicine, Karolinska Institutet, Stockholm, Sweden.
Charlotte
C
Thålin
Department of Clinical Sciences, Danderyd Hospital, Division of Internal Medicine, Karolinska Institutet, Stockholm, Sweden.
Maud
M
Daleskog
Department of Clinical Sciences, Danderyd Hospital, Department of Anesthesia and Intensive Care, Karolinska Institutet, Stockholm, Sweden.
Sophie Paues
SP
Göransson
Department of Biology, Boston University, Boston, MA, 02215, USA.
Daphne
D
Schatzberg
Stress Research Institute, Stockholm University, Stockholm, Sweden.|Department of Clinical Neuroscience, Division of Psychology, Karolinska Institutet, Solna, Stockholm, Sweden.
Julie
J
Lasselin
Department of Clinical Sciences, Danderyd Hospital, Division of Internal Medicine, Karolinska Institutet, Stockholm, Sweden.
Ann-Charlotte
AC
Laska
Department of Clinical Chemistry, Karolinska University Hospital, Stockholm, Sweden.
Anders
A
Kallner
Department of Medical Biochemistry and Biophysics, Division of Translational Medicine and Chemical Biology, Karolinska Institutet, Science for Life Laboratory, Stockholm, Sweden.
Thomas
T
Helleday
Department of Clinical Sciences, Danderyd Hospital, Division of Cardiovascular Medicine, Karolinska Institutet, Stockholm, Sweden.
Håkan
H
Wallén
Department of Clinical Sciences, Danderyd Hospital, Division of Internal Medicine, Karolinska Institutet, Stockholm, Sweden. melanie.demers@ki.se.
Mélanie
M
Demers
D015415:Biomarkers / Q000378:metabolism; D000076302:Citrullination; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D065206:Extracellular Traps / Q000378:metabolism*; D005240:Feasibility Studies; D006657:Histones / Q000378:metabolism*; D006801:Humans; D007249:Inflammation / Q000175:diagnosis*; D008070:Lipopolysaccharides / Q000276:immunology; D009504:Neutrophils / Q000276:immunology*; D015588:Observer Variation; D010949:Plasma / Q000378:metabolism*; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases / Q000378:metabolism; D012680:Sensitivity and Specificity
D015415:Biomarkers; D006657:Histones; D008070:Lipopolysaccharides; C000629235:PADI4 protein, human; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases
Elisa; H3Cit; Human plasma; LPS-induced inflammation; NETs; PAD4