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The quantification of four distinct proteins (α-synuclein, β-amyloid 1-42 , 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-amyloid 42 , 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-amyloid 42  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.

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