Vol.23 No.4

Original Article

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

Authors

Akihito Ishigami1 , Yoshiaki Uchida2 , Tsuyoshi Miyazaki3 , Setsuko Handa1 , Eun-Kyoung Choi4 , Yong-Sun Kim4 , Yasushi Kasahara1 , Naoki Maruyama1

  • Molecular Regulation of Aging, Tokyo Metropolitan Institute of Gerontology (TMIG), 35-2 Sakae-cho, Itabashi-ku, Tokyo 173-0015, Japan
  • Research and Development Division, Fujirebio Inc., Tokyo 192-0031, Japan
  • Department of Geriatric Medicine, Tokyo Metropolitan Institute of Gerontology, Tokyo 173-0015, Japan
  • 1Ilsong Institute of Life Science, Hallym University, Anyang, Gyeonggi-do, 431-060, Korea
Received:

18 May 2012

Accepted:

10 August 2012

Published online:

18 September 2012

Full Text

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Abstract

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 enzymelinked 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 (%ABST%.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.

Key words

Anti-CCP antibody, Pad4, Rheumatoid arthritis, RA diagnosis, Sandwich ELISA