Background and Objectives Antibodies against citrullinated peptides (ACPA) are a hallmark of rheumatoid arthritis (RA) patients and are associated with poor outcome. ACPA are usually detected using the anti-CCP test, identifying antibodies against a broad spectrum of citrullinated antigens. Most citrullinated antigens however appear to be bystander antigens that are not thought to be involved in the disease process. While controversy remains over the identity of the pathogenic antigen, it has been hypothesised that the fine-specificity of the ACPA response in the patient harbours prognostic properties. For this purpose sera will be studied from early RA patients using a novel technique that might reveal differences in that fine-specificity of the ACPA response.
Materials and Methods We recently showed that the antibody response to a particular antigen results in rearranged immunoglobulin segments that are shared between individuals exposed to that antigen. In patients with paraneoplastic neurological syndromes, such sequences were found after proteomics analysis of affinity enriched immunoglobulins. In the current study, ACPA positive RA sera were drawn from 58 patients participating in the tREACH-study. This is a study with a protocolised treatment regime for patients with early RA aimed at obtaining low-disease activity (DAS < 2.4). Poor outcome was defined as the need for treatment with anti-TNF to control disease activity. Sera were affinity enriched using CCP2 ELISA plates, and the specific IgG was analysed by LC-MS.
Results Up to 1 µg of CCP specific IgG was obtained from 180 µl of serum. The LC-MS data was analysed for correlations between ACPA derived peptides and the need for treatment with anti-TNF to control disease activity. However, no such correlations were found in excess of the false discovery rate in this dataset. In addition to ACPA, we also affinity purified rheumatoid factors from sera as a control. These preparations could be distinguished from the ACPA, suggesting our method performed appropriately.
Conclusions Our experiments could not show a significant difference between anti-CCP antibodies in early RA sera from patients with different disease outcome. While this could indicate that anti-CCP fine-specificities are not associated with disease outcome, additional experiments are needed to interpret these results. In future work we will compare IgG with known affinity to subclasses of CCP antigens to verify that our technique distinguishes these more subtle differences in epitope specificity. In addition, it will be investigated whether fine specificity may affect the progression to arthritis in ACPA+ arthralgia patients.
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