Autoantibodies against citrullinated proteins are found in 64-89% of rheumatoid arthritis (RA) patients, with 88-99% specificity. To this end, we report the first unbiased and comprehensive profiling of serum antibodies in RA patients against the human proteome, including the citrullinome and the homocitrullinome using a high-density peptide array. From this data, we identified novel RA specific epitopes and could form the basis of a new RA diagnostic assay.
Our high-density peptide array, comprised of over 4.6 million peptides, contained the entire annotated human proteome. The 20,246 proteins were represented as overlapping 16-mer peptides. In addition to native peptides, citrullinated and homocitrullinated peptides were also included, substituting for arginine and lysine, and provided a comprehensive screen against all possible epitopes.
26 serum samples (8 controls and 18 RA) were profiled on the high-density peptide array. Using RA specific epitopes, we constructed an 8-epitope diagnostic biomarker on the Gyros xPlore instrument with a cohort of 92 serum samples (29 controls and 63 RA), yielding 96.6% specificity and 92.1% sensitivity. The diagnostic biomarker was further validated with an independent cohort of 181 serum samples (54 controls and 127 RA), yielding 94.4% specificity and 85% sensitivity. In both cohorts, the performance of the 8-epitope diagnostic biomarker compared favorably against the Abnova CCP2 assay.
Comprehensive antibody profiling using high-density peptide array not only showed novel RA specific epitopes but also allowed us to construct a novel diagnostic biomarker that is as specific as and more sensitive than the Abnova CCP2 assay.