Molecular Regulation of Aging, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo 173-0015, Japan.
Mod Rheumatol. 2013 Jul;23(4):794-803. doi: 10.1007/s10165-012-0748-0. Epub 2012 Sep 18.
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.
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.
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.
The combination of these sandwich ELISA methods may be a potentially beneficial clinical tool for diagnosing RA.
肽基精氨酸脱亚氨酶 4（PAD4）基因和 PAD4 自身抗体与类风湿关节炎（RA）及其发病机制有关。因此，能够准确测定这些患者外周血中 PAD4 水平的方法将是一种有诊断价值的工具。本研究的目的是改编酶联免疫吸附测定（ELISA）方法，以评估人血液中 PAD4 的水平。
我们制备了重组人（h）PAD1、-2、-3 和 -4 蛋白，以开发针对 hPAD4 的小鼠单克隆抗体。然后，我们生成了针对 hPAD4 的 6 种单克隆抗体，并开发了两种新的夹心 ELISA 方法，用于评估 32 例 RA 患者、10 例骨关节炎患者和 20 名健康个体外周血中的 hPAD4 和 PAD4 自身抗体。
确定了 hPAD4 在患者血浆中的分布。鉴定出两组人群：一组 hPAD4 水平较高（>0.57ng/ml），另一组 hPAD4 水平接近零（<0.1ng/ml）。大多数 hPAD4 水平接近零的患者存在 PAD4 自身抗体。相比之下，大多数具有较高血浆 hPAD4 水平的患者没有检测到 PAD4 自身抗体。
这些夹心 ELISA 方法的组合可能是诊断 RA 的一种潜在有益的临床工具。
