ANALYTICAL PERFORMANCE CHARACTERISTICS OF A PROTOTYPE IMMUNOASSAY FOR QUANTIFICATION OF ALPHA-SYNUCLEIN IN HUMAN CEREBROSPINAL FLUID

Abstract

α-Synuclein is a pre-synaptic protein whose abnormal aggregation is the main characteristic of a sub-group of neurodegenerative disorders called α-synucleinopathies, including but not limited to Parkinson's disease (PD) and dementia with Lewy bodies (DLB). CFS α-Synuclein levels reflect the presence of Lewy bodies in the brain and have been shown to be decreased in PD and DLB patients as compared to healthy aging controls. Robust assays with good lot-to-lot consistency are required for integration into clinical trials. This study documents the analytical performance characteristics of a novel colorimetric ELISA targeting α-Synuclein in biological fluids, with a focus on cerebrospinal fluid (CSF). α-Synuclein concentrations have been determined using a colorimetric ELISA designed and developed by ADx NeuroSciences using two monoclonal antibodies. The assay was qualified for CSF, but can be modified for application for EDTA-plasma samples. After having performed familiarization runs and some adaptation of the test procedures to the needs of a service provider, the assay was challenged internally for its analytical performance characteristics such as precision, parallelism, spike-recovery, working range, sample stability, and interference using commercially available CSF samples (n=40). α-Synuclein is measured in undiluted CSF and 10–50-fold diluted EDTA-plasma using a 3-hour incubation period for the samples. The new assay format specifically quantifies α-Synuclein in the absence of matrix interference as verified after dilution in α-Synuclein low CSF. Spike/recovery testing revealed good recovery rates (93 % ± 7 %). Our internal precision experiments with CSF resulted in an intra- and inter-assay variability (% CV) below 12 % and 11 %, respectively. The working range is confirmed between 90 pg/mL (Lower Limit of Quantitation) to 6142 pg/mL (Upper Limit of Quantitation). The stability of CSF α-Synuclein was assessed with respect to freeze/thawing and short-term stability at room temperature. No major difference with the reference condition was observed. The newly developed α-Synuclein colorimetric ELISA assay meets all of our internal acceptance criteria for clinical trial sample testing and represents an additional tool for patient enrichment in clinical trials or treatment follow-up.