Decrease in Plasma Levels of α-Synuclein Is Evident in Patients with Parkinson’s Disease after Elimination of Heterophilic Antibody Interference

There is substantial biochemical, pathological, and genetic evidence that α-synuclein (A-syn) is a principal molecule in the pathogenesis of Parkinson disease (PD). We previously reported that total A-syn levels in cerebrospinal fluid (CSF), measured with the specific enzyme-linked immunosorbent assay (ELISA) developed by ourselves, were decreased in patients with PD, and suggested the usefulness of A-syn in CSF and plasma as a biomarker for the diagnosis of PD. After our report, a considerable number of studies have investigated the levels A-syn in CSF and in blood, but have reported inconclusive results. Such discrepancies have often been attributed not only to the use of different antibodies in the ELISAs but also to interference from hemolysis. In this study we measured the levels of A-syn in CSF and plasma by using our own sandwich ELISA with or without heterophilic antibody (HA) inhibitor in 30 patients with PD and 58 age-matched controls. We thereby revealed that HA interfered with ELISA measurements of A-syn and are accordingly considered to be an important confounder in A-syn ELISAs. HA produced falsely exaggerated signals in A-syn ELISAs more prominently in plasma samples than in CSF samples. After elimination of HA interference, it was found that hemolysis did not have a significant effect on the signals obtained using our A-syn ELISA. Furthermore, plasma levels of A-syn were significantly lower in the PD group compared with the control group following elimination of HA interference with an HA inhibitor. Our results demonstrate that HA was a major confounder that should be controlled in A-syn ELISAs, and that plasma A-syn could be a useful biomarker for the diagnosis of PD if adequately quantified following elimination of HA interference.

现有大量生物化学、病理学及遗传学证据表明，α-突触核蛋白（α-synuclein）是帕金森病（Parkinson disease, PD）发病机制中的核心致病分子。本团队此前曾利用自主研发的特异性酶联免疫吸附试验（enzyme-linked immunosorbent assay, ELISA）检测脑脊液（cerebrospinal fluid, CSF）中的总α-突触核蛋白水平，发现帕金森病患者的该指标显著降低，并提出脑脊液及血浆中的α-突触核蛋白可作为帕金森病诊断的潜在生物标志物。自本团队的研究发表以来，已有大量研究探讨脑脊液与血液中的α-突触核蛋白水平，但所得结果均未达成一致结论。此类结果差异通常不仅归因于不同酶联免疫吸附试验所使用抗体的差异，还与溶血样本带来的检测干扰有关。本研究纳入30名帕金森病患者与58名年龄匹配的健康对照者，采用自主研发的夹心酶联免疫吸附试验，分别添加或不添加异嗜性抗体（heterophilic antibody, HA）抑制剂，检测两组受试者脑脊液与血浆中的α-突触核蛋白水平。研究结果显示，异嗜性抗体可干扰α-突触核蛋白的酶联免疫吸附试验检测，是该检测方法中重要的混杂因素；且异嗜性抗体对血浆样本中α-突触核蛋白检测信号的虚假放大效应，较脑脊液样本更为显著。在排除异嗜性抗体的干扰后，溶血并未对本研究采用的α-突触核蛋白酶联免疫吸附试验的检测信号产生显著影响。进一步分析显示，在通过异嗜性抗体抑制剂消除干扰后，帕金森病患者的血浆α-突触核蛋白水平显著低于健康对照组。本研究证实，异嗜性抗体是α-突触核蛋白酶联免疫吸附试验中需重点控制的混杂因素；若能通过消除异嗜性抗体干扰实现准确定量，血浆α-突触核蛋白可成为帕金森病诊断的有效生物标志物。