Two novel sandwich ELISAs identify PAD4 levels and PAD4 autoantibodies in patients with rheumatoid arthritis

Objective The peptidylarginine deiminase 4 (PAD4) gene and PAD4 autoantibodies have been associated with rheumatoid arthritis (RA) and its pathogenesis. Therefore, methods for accurately determining their levels in the peripheral blood of these patients would be a diagnostic asset. The objective of our study was to adapt the enzyme-linked immunosorbent assay (ELISA) method for evaluating PAD4 levels in human blood. Methods We prepared recombinant human (h)PAD1, -2, -3, and -4 proteins to develop mouse monoclonal antibodies specific to hPAD4. We then generated six monoclonal antibodies against hPAD4 and developed two new sandwich ELISA methods for evaluating hPAD4 and PAD4 autoantibodies in the peripheral blood from 32 patients with RA, ten patients with osteoarthrosis, and 20 healthy individuals. Results The distribution of hPAD4 in the patients’ plasma was determined. Two populations were identified: one group with high hPAD4 levels (>0.57 ng/mL) and a second group with near-zero levels (<0.1 ng/mL). Most patients approximating zero hPAD4 levels had PAD4 autoantibodies. In contrast, most of those with higher plasma hPAD4 levels did not have detectable PAD4 autoantibodies. Conclusion The combination of these sandwich ELISA methods may be a potentially beneficial clinical tool for diagnosing RA.