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Extended report
Anticitrullinated protein/peptide antibody multiplexing defines an extended group of ACPA-positive rheumatoid arthritis patients with distinct genetic and environmental determinants
  1. Johan Rönnelid1,
  2. Monika Hansson2,
  3. Linda Mathsson-Alm1,3,
  4. Martin Cornillet4,
  5. Evan Reed2,
  6. Per-Johan Jakobsson2,
  7. Lars Alfredsson5,
  8. Rikard Holmdahl6,
  9. Karl Skriner7,
  10. Guy Serre4,
  11. Karin Lundberg2,
  12. Lars Klareskog2
  1. 1 Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden
  2. 2 Department of Medicine, Rheumatology Unit, Karolinska Institutet, Stockholm, Sweden
  3. 3 Thermo Fisher Scientific, Uppsala, Sweden
  4. 4 Laboratory of Epithelial Differentiation and Rheumatoid Autoimmunity, U1056 Inserm, Toulouse University, Toulouse, France
  5. 5 Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden
  6. 6 Department of Medical Inflammation Research, Karolinska Institutet, Stockholm, Sweden
  7. 7 Department of Medicine, Charité University Hospital, Berlin, Germany
  1. Correspondence to Dr Johan Rönnelid, Department of Immunology, Genetics and Pathology, Uppsala University, SE-75185 Uppsala, Sweden; johan.ronnelid{at}


Introduction The second generation anticycliccitrullinated peptide (anti-CCP2) assay detects the majority but not all anticitrullinated protein/peptide antibodies (ACPA). Anti-CCP2-positive rheumatoid arthritis (RA) is associated with HLA-DRB1* shared epitope (SE) alleles and smoking. Using a multiplex assay to detect multiple specific ACPA, we have investigated the fine specificity of individual ACPA responses and the biological impact of additional ACPA reactivity among anti-CCP2-negative patients.

Methods We investigated 2825 patients with RA and 551 healthy controls with full data on anti-CCP2, HLA-DRB1* alleles and smoking history concerning reactivity against 16 citrullinated peptides and arginine control peptides with a multiplex array.

Results The prevalence of the 16 ACPA specificities ranged from 9% to 58%. When reactivity to arginine peptides was subtracted, the mean diagnostic sensitivity increased by 3.2% with maintained 98% specificity. Of the anti-CCP2-negative patients, 16% were found to be ACPA positive. All ACPA specificities associated with SE, and all but one with smoking. Correction for arginine reactivity also conveyed a stronger association with SE for 13/16 peptides. Importantly, when all ACPA specificities were analysed together, SE and smoking associated with RA in synergy among ACPA positive, but not among ACPA-negative subjects also in the anti-CCP2-negative subset.

Conclusions Multiplexing detects an enlarged group of ACPA-positive but anti-CCP2-negative patients with genetic and environmental attributes previously assigned to anti-CCP2-positive patients. The individual correction for arginine peptide reactivity confers both higher diagnostic sensitivity and stronger association to SE than gross ACPA measurement.

  • ant-ccp
  • autoantibodies
  • rheumatoid arthritis

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  • Handling editor Tore K Kvien

  • Contributors JR had full access to all data, is responsible for data integrity, made statistical calculations and drafted the manuscript. JR, MH, LM-A and LK conceived the study. LA provided epidemiological data and statistical expertise. MH and LM-A participated in the development of the ISAC microarray and performed the laboratory work on the EIRA cohort. MC, ER, P-JJ, RH, KS, GS and KL provided peptides for the analyses, including validation of their performance. All authors read, commented on and approved the final manuscript.

  • Competing interests The project is part of the Innovative Medicines Initiative (IMI) project Be The Cure where Karolinska Institutet is a scientific partner and Thermo Fisher Scientific is a commercial partner. The project follows the rules for IMI projects. JR has obtained reagents from Thermo Fisher Scientific for the investigation on other rheumatology cohorts. LM-A is employed by Thermo Fisher Scientific. LK and RH were cofounders of a company, Curara AB, which has previously collaborated with Thermo Fisher Scientific concerning certain technical aspects of the multiplex assay. This development is done with partial support from an ERC Proof of Concept Grant (pRActice) to LK. RH is a coinventor of a patent (US Patent 7148020) protecting the use of the CitC1 and C1 peptides. KS is coinventor of the patents US 13/141,960 and EP 09799354.7 describing the diagnostic use of the hnRNP-A3 peptide epitopes. GS is coinventor of several international patents about ACPA antigens held by BioMérieux Cy and licenced to Eurodiagnostica Cy and Axis-Shield Cy for commercialisation of the CCP2 assays; according to French laws, he receives a part of the royalties paid to the Toulouse III University and the University Hospital of Toulouse. KL is coinventor of patent US12/524,465, describing the diagnostic use of the CEP-1 epitope. The other authors declare that they have no competing interests.

  • Ethics approval The ethics board in Stockholm.

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Data sharing statement All relevant data are available in the manuscript and supplementary files.