Seropositivity and Antibody Profiling of Patients Are Dramatically Impacted by the Features of Peptides Used as Immunosorbents: A Lesson from Anti–Citrullinated Protein/Peptide Antibody

Abstract

Quantification of Abs toward a single epitope is critical to understanding immunobiological processes. In autoimmunity, the prognostic value of the serological profiles of patients draws much attention, but the detection of Abs toward a single epitope is not well controlled. Particularly, the rheumatoid arthritis (RA)–specific anti–citrullinated protein/peptide Abs (ACPA) are specific to a two-atom change on arginyl residues and are considered a heterogeneous family of Abs. As a model, we studied ACPA to decipher how peptide features used as immunosorbent impact Ab detection. We synthesized 30 peptides encompassing immunodominant epitopes of citrullinated fibrin differing by their length and biotin location and tested them using ELISA with 120 sera from RA and non-RA rheumatic disease controls, generating over 3000 experimental measurements. We showed that minor molecular changes in peptide chemical structure had dramatic consequences. Even when peptides exhibited the same epitope, measured Ab titers were extremely variable, and patients’ seropositivity was discordant in up to 50% of cases. The distance between epitope and biotin was the most critical parameter for efficient Ab detection irrespective of biotin position or peptide length. Finally, we identified a 15-mer peptide bearing a single citrullinated epitope detecting almost all ACPA-positive sera, thus revealing a high degree of homogeneity in RA autoimmune response. This integrative analysis deciphers the dramatic impact of the molecular design of peptide-based technologies for epitope-specific Ab quantification. It provides a model for assay development and highlights that the studies using such technologies can give a wrong perception of biological processes and therefore that medical use of data must be cautious.

Are antibodies to fine specificities of citrullinated peptides/proteins useful for stratification of rheumatoid arthritis patients?

“…Indeed, we showed that minor changes in peptide structure dramatically impacted ELISA results. 13 This is confirmed in the present work showing that the Nt-biotinylation of b60-74Cit and the cyclisation of CEP-1 considerably enhance ACPA capture. This underlines the difficulty to clearly define ACPA fine specificities and thus highlights the relativity of final conclusions concerning ACPA profiles.…”

“…Antibodies towards the biotinylated form of the b60-74 peptide (b60-74CitBiotNt), AhFibA and anti-CCP2 antibodies (Immunoscan RA; Euro Diagnostica, Arnhem, The Netherlands) were tested as previously described. 12,13…”

“…12 Moreover, we demonstrated that perceived levels of ACPA in serum towards a given peptide are dramatically impacted by the features of the peptide. 13 Thus, results can highly differ depending on tests used for ACPA profiling, and impact diagnostic sensitivities, correlations of antibody levels and patients' stratification. In the present study, we tackle these questions by testing RA and controls patients with the most commonly tested peptides, analysing their relationships in patients' stratification.…”

“…We previously showed that most sera containing antibodies towards in vitro citrullinated human fibrinogen (AhFibA) or anti‐CCP2 antibodies recognise a single immunodominant fibrin‐derived β60‐74 peptide 12 . Moreover, we demonstrated that perceived levels of ACPA in serum towards a given peptide are dramatically impacted by the features of the peptide 13 . Thus, results can highly differ depending on tests used for ACPA profiling, and impact diagnostic sensitivities, correlations of antibody levels and patients' stratification.…”

In Rheumatoid Arthritis Patients, HLA-DRB1*04:01 and Rheumatoid Nodules Are Associated With ACPA to a Particular Fibrin Epitope

“…However in the present study, the peptides were used biotinylated and linked to Avidin versus immunoadsorbed on solid phase in the pilot study. Such an increased reactivity when using biotinylated peptides, depending on a different presentation of the peptides, was clearly demonstrated with the peptide β60-74cit in a recently published study ( 33 ).…”