Background/Purpose: Anti-citrullinated protein antibodies (ACPA) are highly specific for the diagnosis of patients with RA. However, the predominant B cell epitopes have not yet been defined. The aim of this study was to examine in parallel the individual ACPA antibodies against different peptides derived from citrullinated proteins and investigate whether these antibodies constitute a homogenous population.

Methods: Sera from patients with RA (n = 141), systemic lupus erythematosus (n = 60), Sjögren’s syndrome (n = 54), and healthy controls (n = 100) were tested for their reactivity against 6 citrullinated peptides (pep#2-pep#7) derived from peptidyl arginine deiminase (PAD), vimentin, alpha-enolase, fibrin, type II collagen and filaggrin respectively. A non citrullinated-control peptide derived from PAD was used as control (pep#1). Antibody reactivity was evaluated for each individual peptide by ELISA. Third generation anti-cyclic citrullinated peptide (anti-CCP3) antibodies were also determined. Specificity and sensitivity for anti-peptide antibody were tested by homologous and cross inhibitions assays. Cross reactivity between anti-peptide antibodies was evaluated, after affinity purification, by cross-inhibitions assays.

Results:

Sera from patients with RA reacted diversely with the six citrullinated peptides. More specifically, pep#2 PAD (210-230aa) displayed 29.08% sensitivity, pep#3 vimentin (60-75aa) 29.08%, pep#4 alpha-enolase (5-21aa) 37.59%, pep#5 fibrin (617-631aa) 31.21%, pep#6 type II collagen (358-375aa) 29.97% and pep#7 filaggrin (306-324aa) 28.37% while control pep#1 PAD (621-640aa) showed no reactivity. All reactive peptides were found to be specific for RA (specificity: 91.59%, 93.93%, 95.33%, 95.79%, 97.20% and 97.73% respectively). The sensitivity of anti-CCP3 antibodies and antibodies against equamolar peptide mixture (containing six citrullinated peptides) for RA was 60.78% and 46.08% respectively and specificity 94.12% and 82.22%. Minimal cross reactivity between antibodies against the majority of citrullinated peptides was observed, ranging from 5.43% to 44.98%. A notable cross reaction (>60%) was observed between antibodies to pep#2 and antibodies to other peptides.

Conclusion: This study showed that ACPA in RA, using as substrates peptides from different citrullinated proteins, constitute a heterogeneous population with limited cross reactivity and without a predominant epitope. Studies to evaluate the significance of these findings are now under study in our laboratory.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/fine-specificity-of-anti-citrullinated-peptide-antibodies-discloses-a-heterogeneous-antibody-population-in-rheumatoid-arthritis-ra/