Department of Orthopaedic Surgery, Kyushu University, Fukuoka, Japan.
Division of Host Defense, Medical Institute of Bioregulation, Kyushu University, Fukuoka; Kondo Clinic for Orthopaedics and Rheumatology, Fukuoka; Clinical Research Institute, National Hospital Organization Kyushu Medical Center, Fukuoka, Japan.
Clin Exp Rheumatol. 2018 Jul-Aug;36(4):612-618. Epub 2018 Feb 14.
Since the presence of IgM antibodies is a hallmark of ongoing immune response, we aimed to identify immunologically active rheumatoid arthritis (RA) patients by detecting IgM anti-citrullinated protein antibody (ACPA) levels.
IgM ACPA levels were determined in the serum of 176 RA patients by enzyme-linked immunosorbent assay, in which parameters of reactivity against citrullinated and non-citrullinated peptides were compared to ensure the specificity. Influence of IgM rheumatoid factor (RF) on IgM ACPA detection was examined by removing IgG, using protein G-conjugated beads, or by purifying ACPA, using citrullinated peptide-conjugated beads.
Although IgM specific for citrullinated proteins was detected in some patients (11%), IgM molecules reactive to both citrullinated and non-citrullinated peptides were detected in a substantial number of patient samples (12%). IgM ACPA-positive reactions were associated with the presence of IgG ACPA and IgM RF. Surprisingly, protein G-mediated removal of IgG from the serum eliminated positivity for IgM ACPA, suggesting that IgG ACPA-IgM RF complex was being detected. This assumption was confirmed by the detection of IgM RF in the eluate of protein G beads and citrullinated peptide-conjugated beads.
In an attempt to detect IgM ACPA, we mostly revealed false positive reactions due to the presence of IgM molecules, which were not specific for citrullinated proteins, and IgG ACPA-IgM RF immune complex. The latter complex had been proposed to play a role in the pathogenesis of RA, and here, for the first time, we have demonstrated its presence in the sera of RA patients.
由于 IgM 抗体的存在是持续免疫反应的标志，我们旨在通过检测 IgM 抗瓜氨酸化蛋白抗体（ACPA）水平来识别免疫活性类风湿关节炎（RA）患者。
通过酶联免疫吸附试验（ELISA）检测 176 例 RA 患者血清中的 IgM ACPA 水平，比较针对瓜氨酸化和非瓜氨酸化肽的反应性参数，以确保特异性。通过使用蛋白 G 偶联珠去除 IgG 或使用瓜氨酸化肽偶联珠纯化 ACPA 来检查 IgM 类风湿因子（RF）对 IgM ACPA 检测的影响。
虽然在一些患者（11%）中检测到针对瓜氨酸化蛋白的 IgM 特异性，但在大量患者样本中检测到对瓜氨酸化和非瓜氨酸化肽均有反应的 IgM 分子（12%）。IgM ACPA 阳性反应与 IgG ACPA 和 IgM RF 的存在相关。令人惊讶的是，从血清中去除 IgG 的蛋白 G 介导消除了 IgM ACPA 的阳性反应，这表明检测到 IgG ACPA-IgM RF 复合物。通过检测蛋白 G 珠和瓜氨酸化肽偶联珠洗脱液中的 IgM RF 证实了这一假设。
在试图检测 IgM ACPA 时，我们主要揭示了由于存在非瓜氨酸化蛋白特异性的 IgM 分子和 IgG ACPA-IgM RF 免疫复合物而导致的假阳性反应。有人提出后者复合物在 RA 的发病机制中起作用，在这里，我们首次证明其存在于 RA 患者的血清中。
