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Recognition of citrullinated and carbamylated proteins by human antibodies: specificity, cross-reactivity and the ‘AMC-Senshu’ method
  1. Jing Shi1,
  2. Annemiek Willemze1,
  3. George M C Janssen2,
  4. Peter A van Veelen2,
  5. Jan Wouter Drijfhout2,
  6. Anthony Cerami3,
  7. Tom W J Huizinga1,
  8. Leendert A Trouw1,
  9. René E Toes1
  1. 1Department of Rheumatology, Leiden University Medical Center, Leiden, The Netherlands
  2. 2Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands
  3. 3Department of Nephrology, Leiden University Medical Center, Leiden, The Netherlands
  1. Correspondence to Dr L A Trouw, Department of Rheumatology, C1R, Leiden University Medical Center, Postbus 9600, Leiden 2300 RC, The Netherlands; L.A.Trouw{at}lumc.nl

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Recently, a novel family of autoantibodies in rheumatoid arthritis (RA) patients was described: anti-carbamylated protein (Anti-CarP) antibodies, which target carbamylated (homocitrulline-containing) epitopes.1 ,2 Since citrulline and homocitrulline have a similar structure, we wished to determine to what extent human autoantibodies can differentiate between them. Unlike human antibodies, the anti-modified citrulline (AMC) antibody developed by Dr Senshu3–8 is able to recognise citrullinated epitopes irrespective of the neighbouring amino acids. Thus, we also aimed to verify whether the AMC assay could distinguish between these two amino acids.

To address this, we loaded gels with citrullinated, carbamylated and non-modified forms of foetal calf's serum (FCS) and human fibrinogen (Fib). Gels loaded with equal amounts of these protein preparations (figure 1A) were used for western blotting and staining with the ‘AMC-Senshu’ method. Both the citrullinated and the carbamylated forms of the proteins tested were strongly recognised, whereas, the non-modified form did not reveal any staining (figure 1B). Staining similar western blots with selected human sera1 revealed that sera-positive …

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