Ishii Ryotaro, Tokuda Takahiko, Tatebe Harutsugu, Ohmichi Takuma, Kasai Takashi, Nakagawa Masanori, Mizuno Toshiki, El-Agnaf Omar M A
Department of Neurology, Kyoto Prefectural University of Medicine, Kyoto, Japan.
Department of Neurology, Kyoto Prefectural University of Medicine, Kyoto, Japan; Department of Molecular Pathobiology of Brain Diseases, Kyoto Prefectural University of Medicine, Kyoto, Japan.
PLoS One. 2015 Apr 7;10(4):e0123162. doi: 10.1371/journal.pone.0123162. eCollection 2015.
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.
有大量生物化学、病理学和遗传学证据表明，α-突触核蛋白（A-syn）是帕金森病（PD）发病机制中的主要分子。我们之前报道，使用我们自己开发的特异性酶联免疫吸附测定（ELISA）法测量，帕金森病患者脑脊液（CSF）中的总A-syn水平降低，并提示脑脊液和血浆中的A-syn作为帕金森病诊断生物标志物的有用性。在我们的报告之后，大量研究调查了脑脊液和血液中A-syn的水平，但报告结果尚无定论。这种差异通常不仅归因于ELISA中使用了不同抗体，还归因于溶血的干扰。在本研究中，我们使用自己的夹心ELISA法，在有或没有嗜异性抗体（HA）抑制剂的情况下，测量了30例帕金森病患者和58例年龄匹配对照者脑脊液和血浆中A-syn的水平。我们由此发现，HA干扰了A-syn的ELISA测量，因此被认为是A-syn ELISA中的一个重要混杂因素。HA在A-syn ELISA中产生的假阳性信号在血浆样本中比在脑脊液样本中更明显。消除HA干扰后，发现溶血对使用我们的A-syn ELISA获得的信号没有显著影响。此外，在使用HA抑制剂消除HA干扰后，帕金森病组的血浆A-syn水平与对照组相比显著降低。我们的结果表明，HA是A-syn ELISA中应控制的主要混杂因素，并且如果在消除HA干扰后进行充分定量，血浆A-syn可能是帕金森病诊断的有用生物标志物。
