Background/Purpose: Anti-citrullinated protein/peptide autoantibodies (ACPAs) define a major subset of rheumatoid arthritis (RA) patients. Cyclic citrullinated peptides (CCP) are commonly used as the antigen for assessing ACPAs. However, these synthetic peptides may not represent the citrullinated proteins found in vivo. We have utilized ECV304, a human cell line containing high protein arginine deiminases (PAD) levels, to generate a naturally derived citrullinome. Next, we assessed by proteomics the ECV304 antigens recognized by antibodies present in RA sera. Finally, we compared the performance of an in house ECV304-based ELISA with that of a commercial anti-CCP2-ELISA for RA diagnosis.

Methods: Citrullination was assessed in ECV 304 and other human cell lines (n=7) by Western blot and the amount of citrulline generated was quantified by a colorimetric assay. PAD isotypes present in ECV304 were determined by PCR. ECV304 citrullinated proteins/peptides were immunoprecipitated by autoantibodies present in RA sera, and characterized by mass spectrometry. The ECV304-based ELISA contained a citrullinated ECV304 cell extract and myelin basic protein (MBP, an arginine-rich potential citrullinated -epitope carrier). The performance of this assay was tested in sera from 1) 74 patients with a clinical diagnosis of RA (55 CCP positive, 50 rheumatoid factor [RF] positive; 8 CCP and RF negative), 2) 51 patients with non-RA rheumatic diseases (non-RA controls), and 3) 25 healthy controls.

Results: Among 7 human cell lines tested, ECV304 was the only one that abundantly citrullinated self-proteins and exogenous MBP. ECV304 expressed PAD2 and PAD3 iso-enzymes, and the ECV304 citrullinated proteins/peptides were detected by autoantibodies present in RA sera. Proteomic analysis confirmed the presence of known citrullinated RA antigens (i.e., calreticulin, profiling 1 and vinculin), as well as novel targets such as: 14-3-3 protein beta/alpha, 14-3-3 protein zeta/delta, mitochondrial peroxiredoxin-5, and transkelotase in the ECV304 extracts. The net binding to ECV304 citrullinated proteins (compared to non-citrullinated proteins) was the ELISA readout, and the cut-off of the test was defined to optimize specificity. The sensitivity, specificity, positive and negative predictive values of the ECV304-based ELISA were 39%, 98%, 97% and 63%, respectively.

Conclusion: The human cell line ECV304 possesses all the molecular machinery to citrullinate self-proteins that are recognized by ACPA-positive RA sera. Due to its high specificity, and low cost, the ECV304-based ELISA could represent an alternative to ACPA tests for the diagnosis of RA in resource-limited settings.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/ecv304-cells-self-citrullinate-proteins-targeted-by-anti-citrullinated-protein-peptide-autoantibodies-from-rheumatoid-arthritis-patients/