Background/Purpose: Autoimmunity in rheumatoid arthritis (RA) is characterized by autoantibodies to citrullinated proteins/peptides (ACPA). These antibodies (present in 60-70% of patients) antedate clinical onset and associate with an erosive disease course, suggesting a direct pathogenic involvement in disease initiation and progression. With this study, we aimed to utilise plasma and synovial fluids (SF) from patients with RA, for the purification of ACPAs reactive with the peptides used in the CCP2 ELISA assay. Furthermore, to characterise their frequency in plasma and SF as well as reactivity with different autoantigen-derived peptides.

Methods: Plasma (n=16) and SF (n=26) samples were collected, with informed consent and ethical approval, from RA patients with anti-CCP2 IgG levels above 300AU/ml. Total IgG was isolated on Protein G columns, and subsequently applied to CCP2 affinity columns, kindly donated by EuroDiagnostica. Flow through and eluate fractions were assayed for antibody responses using the CCP2 ELISA, as well as in-house ELISAs, for analysis of reactivity to citrullinated peptides from a-enolase, vimentin, fibrinogen and collagen type II.

Results: Pure and intact anti-CCP IgG antibodies were efficiently isolated from plasma and SF samples. No citrulline-reactivity was detected in the CCP2 column flow through fractions. Purified anti-CCP IgG from different patients showed differences in binding to CCP2 ELISA plates (assayed at the same antibody concentration), still a majority showed reactivity with the four citrullinated autoantigen-derived peptides. Purified anti-CCP IgG also bound citrullinated, but not uncitrullinated, human fibrinogen, by Western blot, while the corresponding CCP2 column flow through IgG bound neither citrullinated nor uncitrullinated fibrinogen. A median of 1.5% of the IgG pool in plasma and 2.2% in SF, with four SF samples reaching 6%, were CCP2-reactive.

Conclusion: Here we demonstrate an efficient and robust method to isolate anti-CCP IgG from plasma and SF. Furthermore, that ACPAs reactive with epitopes on different citrullinated autoantigens (i.e. a-enolase, vimentin, fibrinogen and collagen type II), which are largely non-cross-reactive, are captured by the CCP2 column. These purified anti-CCP IgG molecules will provide us with new opportunities to investigate functional and structural aspects of human anti-citrullinated protein/peptide antibodies, including pathogenicity.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/affinity-purification-and-characterisation-of-anti-ccp-antibodies-from-plasma-and-synovial-fluids-of-patients-with-rheumatoid-arthritis/