title: Anti-cyclic citrullinated peptide antibodies are a collection of anti-citrullinated protein antibodies and contain overlapping and non-overlapping reactivities.
abstract: OBJECTIVE
  Anti-citrullinated protein antibodies (ACPA) and anti-cyclic citrullinated peptide (anti-CCP) antibodies are a hallmark of rheumatoid arthritis and are believed to play a role in disease pathogenesis. These antibodies are typically detected in ELISA with citrullinated peptides (eg, CCP2) or proteins as antigens. The absolute concentration of anti-CCP antibodies in serum is unknown. Although antibodies to several citrullinated proteins can mainly be detected within anti-CCP-positive sera, it is currently unknown whether anti-CCP antibodies are in fact ACPA. Likewise, it is unknown to what extent antibody responses to different citrullinated antigens are crossreactive.
  
  
  METHODS
  An affinity purification method was established in which citrullinated antigen-specific antibodies were eluted from ELISA plates and then used for detection of other citrullinated antigens in ELISA or western blot. For additional crossreactivity studies, ELISA-based inhibition assays were performed with citrullinated or control peptides as inhibitors.
  
  
  RESULTS
  The concentration of anti-CCP IgG antibodies was estimated to be at least 30 μg/ml in patients with high anti-CCP levels (>1600 μg/ml). Affinity-purified anti-CCP antibodies were able to recognise citrullinated fibrinogen (cit-fib) and citrullinated myelin basic protein (cit-MBP) on western blot. Furthermore, antibodies specific for cit-fib and cit-MBP were crossreactive. However, additional crossreactivity studies indicated that non-overlapping antibody responses to citrullinated peptides can also exist in patients.
  
  
  CONCLUSIONS
  This report shows for the first time that anti-CCP antibodies recognise multiple citrullinated proteins and are thus a collection of ACPA. More importantly, the data indicate that different ACPA responses are crossreactive, but that crossreactivity is not complete, as distinct non-crossreactive responses can also be detected in patients with RA.
authors.affiliation: Department of Rheumatology, Leiden University Medical Center, Leiden, The Netherlands. a.ioan@lumc.nl
authors.forename: Andreea
authors.initials: A
authors.lastname: Ioan-Facsinay
authors.forename: Hanane
authors.initials: H
authors.lastname: el-Bannoudi
authors.forename: Hans U
authors.initials: HU
authors.lastname: Scherer
authors.forename: Diane
authors.initials: D
authors.lastname: van der Woude
authors.forename: Henri A
authors.initials: HA
authors.lastname: Ménard
authors.forename: Maximilien
authors.initials: M
authors.lastname: Lora
authors.forename: Leendert A
authors.initials: LA
authors.lastname: Trouw
authors.forename: Tom W J
authors.initials: TW
authors.lastname: Huizinga
authors.forename: Rene E M
authors.initials: RE
authors.lastname: Toes
mesh_terms: D000915:Antibody Affinity / Q000276:immunology; D001172:Arthritis, Rheumatoid / Q000276:immunology*; D001323:Autoantibodies / Q000097:blood / Q000276:immunology*; D001324:Autoantigens / Q000276:immunology; D002956:Citrulline / Q000276:immunology; D003429:Cross Reactions / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood / Q000276:immunology; D010456:Peptides, Cyclic / Q000276:immunology*
chemical_list: D001323:Autoantibodies; D001324:Autoantigens; D007074:Immunoglobulin G; D010456:Peptides, Cyclic; C487763:cyclic citrullinated peptide; D002956:Citrulline