Background Autoimmunity in rheumatoid arthritis (RA) is characterized by autoantibodies to citrullinated proteins/peptides (ACPA)1. 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 progression2.
Objectives With this study, we aimed to develop an efficient method for the purification of human ACPA, and to characterize their frequency and fine-specificity pattern in synovial fluid (SF) and plasma of RA patients.
Methods SF and plasma samples were collected with informed consent and ethical approval from patients (fulfilling the ACR criteria for RA) with high anti-CCP antibody levels. SF samples (n=36) were first treated with hyaluronidase to decrease viscosity, then proteins were precipitated with ammonium sulfate, dissolved and further dialysed against phosphate buffered saline (PBS), before the IgG fractions were purified on ProteinG columns (GE Healthcare, Uppsala, Sweden). Plasma samples (n=10) were diluted in PBS before applied to the ProteinG column. ACPAs were further purified using CCP2 affinity columns, kindly provided by Euro-Diagnostica. Recovery and purity of total IgG and anti-CCP IgG were analysed using SDS-PAGE, Nanodrop (Thermo Scientific, Wilmington, DE, USA) and the CCP2-ELISA kit. Fine-specificity of the purified ACPAs were investigated using in-house ELISAs, with peptides from citrullinated α-enolase (CEP-1), -vimentin (Cit-vim), -fibrinogen (Cit-fib) and -collagen type II (Cit-C1).
Results Anti-CCP IgG could efficiently be purified from SF and plasma, using ProteinG-, followed by CCP2-, columns. No CCP IgG response could be detected in the flow-through fractions. Higher concentrations of total IgG were found in plasma (13,6 mg/ml) compared to SF (4,2 mg/ml), while a higher percentage of CCP-specific IgG was detected in SF (3%), compared to plasma (2%). The purified anti-CCP IgG fractions cross-reacted with CEP-1, Cit-vim, Cit-fib and Cit-C1, while no reactivity to these citrullinated antigens were detected in the IgG flow-through fractions. Anti-CCP IgG dilution curves (starting at 10 μg/ml of purified antibodies) demonstrated differences in affinity between patients, which may correspond to the different ACPA-fine specificity patterns seen in patients.
Conclusions The described methodology efficiently purifies ACPAs with multiple specificities, which will allow for their use in in vivo and in vitro studies, to further elucidate their arthritogenic and pathogenic capacity. In addition, the ACPAs will be tools for future immunoprecipitation-, immunoblotting- and immunohistochemistry experiments.
Schellekens, G. A. et al., The diagnostic properties of rheumatoid arthritis antibodies recognizing a cyclic citrullinated peptide. Arthritis Rheum 43(1), 155 (2000).
van der Helm-van Mil AH, Verpoort KN, Breedveld FC, Toes RE, Huizinga TW, Antibodies to citrullinated proteins and differences in clinical progression of rheumatoid arthritis. Arthritis Res Ther 7(5), R949 (2005).
Disclosure of Interest None Declared
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