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

Abstract

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.

Fig 1. Calibration curves of recombinant proteins are highly reproducible.

Normalized mean values (± standard deviation) from seven independent experiments performed with multiplex assay plates are presented.

Fig 2. Highly comparable performance in quantification of pooled versus individual recombinant proteins on multiplex assay plates.

Individual as well as pooled recombinant calibrators were serially diluted by a factor of three for analysis. Signal intensities were comparable under both conditions.

Fig 3. Linearity testing for serial dilution of proteins spiked into CSF samples.

Three CSF samples were spiked with approximately 100 times the endogenous concentration of aSyn, DJ-1 and t-tau protein as well as 25 times of endogenous Aβ₄₂ concentration. CSF samples were serially diluted by a factor of two for analysis. Protein concentrations were normalized for the fourth dilution. Relative protein concentrations are presented. A: aSyn, B: Aβ₄₂, C: DJ-1, D: t-tau protein.

Fig 4. Parallelism of analyte quantification in serially diluted CSF samples.

Pooled CSF and three individual CSF samples were serially diluted by a factor of two for analysis. Protein concentrations were normalized for the 1:8 dilutions. A: aSyn, B: Aβ₄₂, C: DJ-1, D: t-tau protein.

Fig 5. Recovery rates of spiked calibrators into individual CSF samples.

Recombinant calibrators were spiked at three different concentrations into four CSF samples. Concentrations of spike solutions and endogenous protein concentrations were determined in parallel. Recovery rates were calculated taking into account both the endogenous CSF concentration and the protein concentration of the spike solutions. A: aSyn, B: Aβ₄₂, C: DJ-1, D: t-tau protein.

Fig 6. Quantification of CSF samples in singleplex versus multiplex assays.

Eight CSF samples from neurological controls and patients with PD were analyzed both in singleplex (except for Aβ₄₂) and (SP) multiplex assays (MP). Results were obtained from non-diluted samples.