Development of a Novel Electrochemiluminescence ELISA for Quantification of α-Synuclein Phosphorylated at Ser(129) in Biological Samples

[Image: see text] Synucleinopathies are a group of neurodegenerative diseases including Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). These diseases are characterized by the aggregation and deposition of α-synuclein (α-syn) in Lewy bodies (LBs) in PD and DLB or as glial cytoplasmic inclusions in MSA. In healthy brains, only ∼4% of α-syn is phosphorylated at Ser(129) (pS(129)-α-syn), whereas >90% pS(129)-α-syn may be found in LBs, suggesting that pS(129)-α-syn could be a useful biomarker for synucleinopathies. However, a widely available, robust, sensitive, and reproducible method for measuring pS(129)-α-syn in biological fluids is currently missing. We used Meso Scale Discovery (MSD)’s electrochemiluminescence platform to create a new assay for sensitive detection of pS(129)-α-syn. We evaluated several combinations of capture and detection antibodies and used semisynthetic pS(129)-α-syn as a standard for the assay at a concentration range from 0.5 to 6.6 × 10(4) pg/mL. Using the antibody EP1536Y for capture and an anti-human α-syn antibody (MSD) for detection was the best combination in terms of assay sensitivity, specificity, and reproducibility. We tested the utility of the assay for the detection and quantification of pS(129)-α-syn in human cerebrospinal fluid, serum, plasma, saliva, and CNS-originating small extracellular vesicles, as well as in mouse brain lysates. Our data suggest that the assay can become a widely used method for detecting pS(129)-α-syn in biomedical studies including when only a limited volume of sample is available and high sensitivity is required, offering new opportunities for diagnostic biomarkers, monitoring disease progression, and quantifying outcome measures in clinical trials.