Development of a Novel Electrochemiluminescence ELISA for Quantification of α-Synuclein Phosphorylated at Ser¹²⁹ in Biological Samples

Abstract

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 Ser129 (pS129-α-syn), whereas >90% pS129-α-syn may be found in LBs, suggesting that pS129-α-syn could be a useful biomarker for synucleinopathies. However, a widely available, robust, sensitive, and reproducible method for measuring pS129-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 pS129-α-syn. We evaluated several combinations of capture and detection antibodies and used semisynthetic pS129-α-syn as a standard for the assay at a concentration range from 0.5 to 6.6 × 104 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 pS129-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 pS129-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.

Rapid FRET Assay for the Early Detection of Alpha-Synuclein Aggregation in Parkinson’s Disease

“…It was found that about 90% of α-Syn was phosphorylated at Ser129 in LBs, while only 4% of α-Syn contains this modification in the normal brain, 12 which indicates that the phosphorylation at Ser129 of α-Syn plays an important role in PD. 13 been carried out; however, the findings were inconsistent. Samuel et al prepared Ser129 phosphorylation (pS129) α-Syn by coexpressing polo-like kinase 2 (PLK-2) and α-Syn in E. coli and found that the pS129 α-Syn promoted in vitro fibrillation.…”

Single-Molecule Protein Analysis by Centrifugal Droplet Immuno-PCR with Magnetic Nanoparticles

“…50,51 For instance, the concentration of phosphorylated α-Syn was reported to be higher in PD patients than in healthy individuals (∼756.8 vs ∼143.4 ng/mL). 52 The low abundance of this protein has made measurement with existing tools challenging, including conventional chemiluminescence and ELISA. 4,53 ■ METHODS AND MATERIALS Reagents and Materials.…”

“…Studies have suggested that serum α-Syn is a biomarker for individuals at the risk of developing PD . All three subtypes (α, β, and γ) of this protein were indicated to have clinical values. , Furthermore, the phosphorylation of α-Syn was a key indicator of the pathological progression of PD. , For instance, the concentration of phosphorylated α-Syn was reported to be higher in PD patients than in healthy individuals (∼756.8 vs ∼143.4 ng/mL) . The low abundance of this protein has made measurement with existing tools challenging, including conventional chemiluminescence and ELISA. , …”

Plasma versus serum for extracellular vesicle (EV) isolation: A duel for reproducibility and accuracy for CNS‐originating EVs biomarker analysis

“…Most studies investigating CNS‐originating EV biomarkers have used serum (Dutta et al, 2021; Dutta, Hornung, Taha, Biggs, et al, 2023; Dutta, Hornung, Taha, & Bitan, 2023; Jiang et al, 2021; Taha et al, 2023; Taha & Ati, 2023) and/or plasma (Fiandaca et al, 2015; Pulliam et al, 2019; Shi et al, 2014; Taha & Ati, 2023) as media for immunoprecipitation of the EVs directly or as part of a two‐step procedure where EVs are first isolated and CNS‐originating EVs are then immunoprecipitated using specific antibodies. However, potential differences between these two media and their implications for downstream analysis have not been thoroughly investigated despite previous studies showing that preanalytical variations can affect the number of larger‐sized and smaller‐sized EVs isolated from blood (Baek et al, 2016; György et al, 2014; Jayachandran et al, 2012; Karimi et al, 2022; Lacroix et al, 2012; Liu et al, 2018; Palviainen et al, 2020; Zhang et al, 2022).…”