A First Tetraplex Assay for the Simultaneous Quantification of Total α-Synuclein, Tau, β-Amyloid42 and DJ-1 in Human Cerebrospinal Fluid

The quantification of four distinct proteins (α-synuclein, β-amyloid1-42, DJ-1, and total tau) in cerebrospinal fluid (CSF) has been proposed as a laboratory-based platform for the diagnosis of Parkinson’s disease (PD) and Alzheimer’s disease (AD). While there is some clinical utility in measuring these markers individually, their usage in routine clinical testing remains challenging, in part due to substantial overlap of concentrations between healthy controls and diseased subjects. In contrast, measurement of different analytes in a single sample from individual patients in parallel appears to considerably improve the accuracy of AD or PD diagnosis. Here, we report the development and initial characterization of a first, electrochemiluminescence-based multiplex immunoassay for the simultaneous quantification of all four proteins (‘tetraplex’) in as little as 50 μl of CSF. In analytical performance experiments, we assessed its sensitivity, spike-recovery rate, parallelism and dilution linearity as well as the intra- and inter-assay variability. Using our in-house calibrators, we recorded a lower limit of detection for α-synuclein, β-amyloid42, DJ-1, and t-tau of 1.95, 1.24, 5.63, and 4.05 pg/ml, respectively. The corresponding, linear concentration range covered >3 orders of magnitude. In diluted CSF samples (up to 1:4), spike-recovery rates ranged from a low of 55% for β-amyloid42 to a high of 98% for DJ-1. Hillslopes ranged from 1.03 to 1.30, and inter-assay variability demonstrated very high reproducibility. Our newly established tetraplex assay represents a significant technical advance for fluid-based biomarker studies in neurodegenerative disorders allowing the simultaneous measurement of four pivotal makers in single CSF specimens. It provides exceptional sensitivity, accuracy and speed.

脑脊液（cerebrospinal fluid, CSF）中四种不同蛋白质（α-突触核蛋白、β-淀粉样蛋白1-42、DJ-1以及总tau）的定量检测，已被提出作为基于实验室的帕金森病（Parkinson’s disease, PD）与阿尔茨海默病（Alzheimer’s disease, AD）诊断平台。尽管单独检测这些标志物具备一定临床应用价值，但在常规临床检验中推广其应用仍面临诸多挑战，部分原因在于健康对照人群与疾病患者的标志物浓度存在显著重叠。相较之下，对单份患者样本中的多种分析物进行并行检测，似乎可显著提升阿尔茨海默病或帕金森病的诊断准确率。本研究报道了首款基于电化学发光（electrochemiluminescence）的多重免疫检测方法的开发与初步表征，该方法可在仅50 μl脑脊液中同时定量上述四种蛋白质（下称“四联检测法”）。在分析性能验证实验中，我们评估了该方法的灵敏度、加标回收率、平行性与稀释线性，以及批内与批间变异度。使用实验室自制校准品，我们测得α-突触核蛋白、β-淀粉样蛋白42、DJ-1以及总tau的检出下限分别为1.95、1.24、5.63与4.05 pg/ml。对应线性浓度范围覆盖超过3个数量级。在稀释后的脑脊液样本（最高稀释比例1:4）中，加标回收率介于β-淀粉样蛋白42的最低值55%至DJ-1的最高值98%之间。斜率范围为1.03至1.30，批间变异度展现出极高的重现性。本研究新建立的四联检测法，为神经退行性疾病的体液生物标志物研究带来了重大技术突破，可在单份脑脊液标本中同时检测四种关键标志物。该方法具备优异的灵敏度、准确度与检测速度。