title: Application of synthetic peptides for detection of anti-citrullinated peptide antibodies.
abstract: Anti-citrullinated protein antibodies (ACPAs) are a hallmark of rheumatoid arthritis (RA) and represent an important tool for the serological diagnosis of RA. In this study, we describe ACPA reactivity to overlapping citrullinated Epstein-Barr virus nuclear antigen-1 (EBNA-1)-derived peptides and analyze their potential as substrates for ACPA detection by streptavidin capture enzyme-linked immunosorbent assay. Using systematically overlapping peptides, containing a 10 amino acid overlap, labelled with biotin C-terminally or N-terminally, sera from 160 individuals (RA sera (n=60), healthy controls (n=40), systemic lupus erythematosus (n=20), Sjögren's syndrome (n=40)) were screened for antibody reactivity. Antibodies to a panel of five citrullinated EBNA-1 peptides were found in 67% of RA sera, exclusively of the IgG isotype, while 53% of the patient sera reacted with a single peptide, ARGGSRERARGRGRG-Cit-GEKR, accounting for more than half of the ACPA reactivity alone. Moreover, these antibodies were detected in 10% of CCP2-negative RA sera. In addition, 47% of the RA sera reacted with two or three citrullinated EBNA-1 peptides from the selected peptide panel. Furthermore, a negative correlation between the biotin attachment site and the location of citrulline in the peptides was found, i.e. the closer the citrulline was located to biotin, the lower the antibody reactivity. Our data suggest that citrullinated EBNA-1 peptides may be considered a substrate for the detection of ACPAs and that the presence of Epstein-Barr virus may play a role in the induction of these autoantibodies.
authors.affiliation: Department of Autoimmunology and Biomarkers, Statens Serum Institut, Artillerivej 5, 2300 Copenhagen S, Denmark.
authors.forename: Nicole Hartwig
authors.initials: NH
authors.lastname: Trier
authors.affiliation: Department of Autoimmunology and Biomarkers, Statens Serum Institut, Artillerivej 5, 2300 Copenhagen S, Denmark.
authors.forename: Bettina Eide
authors.initials: BE
authors.lastname: Holm
authors.affiliation: Department of Rheumatology, Glostrup Hospital, Nordre Ringvej 57, 2600 Glostrup, Denmark.
authors.forename: Ole
authors.initials: O
authors.lastname: Slot
authors.affiliation: Department of Rheumatology, Frederiksberg Hospital, Nordre Fasanvej 57, 2000 Frederiksberg, Denmark.
authors.forename: Henning
authors.initials: H
authors.lastname: Locht
authors.affiliation: Department of Rheumatology, Odense University Hospital, Søndre Boulevard 29, 5000 Odense C, Denmark.
authors.forename: Hanne
authors.initials: H
authors.lastname: Lindegaard
authors.affiliation: Epidemiology, Biostatistics and Bio-demography, Institute of Public Health, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark.
authors.forename: Anders
authors.initials: A
authors.lastname: Svendsen
authors.affiliation: Department of Rheumatology, Copenhagen University Hospital, Rigshospitalet, Blegdamsvej 9, 2100 Copenhagen, Denmark.
authors.forename: Christoffer Tandrup
authors.initials: CT
authors.lastname: Nielsen
authors.affiliation: Department of Rheumatology, Copenhagen University Hospital, Rigshospitalet, Blegdamsvej 9, 2100 Copenhagen, Denmark.
authors.forename: Søren
authors.initials: S
authors.lastname: Jacobsen
authors.affiliation: Department of Rheumatology, Skåne University Hospital, Lund University, S-20502 Malmø, Sweden.
authors.forename: Elke
authors.initials: E
authors.lastname: Theander
authors.affiliation: Department of Autoimmunology and Biomarkers, Statens Serum Institut, Artillerivej 5, 2300 Copenhagen S, Denmark. Electronic address: gh@ssi.dk.
authors.forename: Gunnar
authors.initials: G
authors.lastname: Houen
mesh_terms: D000595:Amino Acid Sequence; D000918:Antibody Specificity; D001172:Arthritis, Rheumatoid / Q000097:blood* / Q000276:immunology; D001323:Autoantibodies / Q000097:blood*; D016022:Case-Control Studies; D002956:Citrulline / Q000097:blood; D004854:Herpesvirus 4, Human / Q000276:immunology; D006801:Humans; D008180:Lupus Erythematosus, Systemic / Q000097:blood / Q000276:immunology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology; D010455:Peptides / Q000737:chemistry; D014764:Viral Proteins / Q000276:immunology
chemical_list: D001323:Autoantibodies; C576376:EBNA 1 protein (35-58), citrullinated-; D010446:Peptide Fragments; D010455:Peptides; D014764:Viral Proteins; D002956:Citrulline
keywords: Citrullinated epitopes; EBNA-1; Peptides; Streptavidin capture ELISA