Background and Objectives Anti-citrullinated protein/peptide antibodies (ACPAs) are the most sensitive and specific serological markers of RA. To identify the optimal epitopes that detect different subgroups of RA patients with high sensitivity and specificity, we have investigated citrulline and arginine containing peptides derived from filaggrin, collagen or vimentin. We have identified a citrulline-containing peptide panel that was recognised by RA sera with high specificity. Our aim was to compare this peptide panel with the conventionally used serological assays and to detect peptide-specific ACPA producing B-cells in in vitro cultures.
Materials and Methods Previously selected citrulline- and arginine-containing filaggrin, vimentin and collagen peptide epitopes were investigated. We compared the recognition of these peptides by RA and control sera using indirect ELISA. B-cells were purified from peripheral blood by negative selection, IgG production was stimulated by B-cell activators (R848 and recombinant human IL-2) provided with the human ELISPOT kit. Antibody producing cells were enumerated after 4 days culture by using peptide-specific ELISPOT assay.
Results Sera samples from 247 RA and 148 age-matched (57 ± 14 years) healthy controls were collected. The citrulline peptide panel detected approximately 80% of RA patients, including 20% of seronegative/CCP negative patients as well. Individual peptides detected different subgroups of RA patients. The more peptides recognised by a particular RA serum sample, the more severe the disease of the patient was. In vitro cultured B-cells from selected RA patients synthesised multiple citrulline-containing peptide-specific antibodies after polyclonal stimulation, while B-cells from healthy blood donors did not.
Conclusions The citrulline peptide panel can detect 20% of ACPA negative RA patients thus may have a prognostic value. Furthermore, the panel is suitable to detect citrulline peptidespecific antibody producing cells, thus enables us to study ACPA producing B-cells of RA patients.
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