Comparison of Different Platform Immunoassays for the Measurement of Plasma Alpha-Synuclein in Parkinson’s Disease Patients

Abstract

Background: The identification of reliable biomarkers in Parkinson’s disease (PD) would provide much needed diagnostic accuracy, a means of monitoring progression, objectively measuring treatment response, and potentially allowing patient stratification within clinical trials. Whilst the assessment of total alpha-synuclein in biofluids has been identified as a promising biomarker, conflicting trends in these levels across patient plasma samples relative to controls has limited its use. Different commercially available assay platforms that have been used to measure alpha-synuclein may contribute to different study outcomes. Objective: To compare different platform immunoassays for the measurement of total alpha-synuclein using the same plasma samples from 49 PD patients and 47 controls. Methods: Total plasma alpha-synuclein concentrations were assessed using the BioLegend, MesoScale Discovery, and Quanterix platform in plasma samples from PD patients and matched controls. Results: A significant increase in total plasma alpha-synuclein was observed in PD patients using the Biolegend (10%), Mesoscale Discovery (13%) and Quanterix (39%) assays. The Mesoscale Discovery and Quanterix assays showed the strongest correlations (r = 0.78, p < 0.0001) with each other, whilst the Quanterix platform demonstrated the lowest variation and highest effect size. Inclusion of age, sex and hemoglobin levels as covariates in the analysis of total alpha-synuclein improved the ability of all three immunoassays to detect a significant difference between patients and controls. Conclusion: All three immunoassays were sensitive enough to detect group level differences between PD patients and controls, with the largest effect size observed with the Quanterix assay. These results may help inform assay choices in ongoing clinical trials.

Plasma α‐synuclein and phosphorylated tau 181 as a diagnostic biomarker panel for de novo Parkinson’s disease

“…The findings that plasma α-syn levels are higher in PD patients compared with HCs, controlling for age and sex, using the ultrasensitive Simoa HD-X platform, are consistent with previous research results (Ng et al, 2019;Youssef et al, 2021). Studies using other platform immunoassays, such as immunomagnetic reduction (IMR), Biolegend, and Mesoscale Discovery, also report elevated plasma α-synuclein in PD patients compared with controls (Chang et al, 2019;Youssef et al, 2021), although there are some conflicting reports using similar platforms (Besong-Agbo et al, 2013;Foulds et al, 2013). In view of our findings, it may be less important to use platforms with higher sensitivity and more important to consider pre-analytical factors, including hemolysis and patients' demographic data.…”

Comparative Analysis of Total Alpha-Synuclein (αSYN) Immunoassays Reveals That They Do Not Capture the Diversity of Modified αSYN Proteoforms

“…Less sensitive platforms might not be able to measure low-abundance species, therefore overlooking and de facto flattening the contribution of these proteins to total ␣SYN levels, compared to measurements obtained by ultrasensitive platforms with a much lower limit of detection. For instance, the increased ␣SYN levels in plasma of PD patients observed with the ultrasensitive Quanterix assay compared to other less sensitive methods could be related to the increased sensitivity of this platform, which may facilitate the detection and quantitation of low-abundance forms of ␣SYN, as discussed recently in [21]. On the other hand, assays with low dynamic ranges of their standard curves and low upper limits of quantification (ULOQ) would have decreased sensitivity and might account for saturation of the signal when certain species are highly abundant in the analyzed matrix.…”

“…This was mainly following the seminal discovery that mutations in the SNCA gene encoding ␣SYN cause familial PD [10,11], and that aggregated and post-translationally modified (PTM) forms of ␣SYN, including phosphorylated, nitrated, ubiquitinated, and truncated species, are key components of Lewy bodies (LB) and Lewy neurites, the main neuropathological hallmark of PD and other synucleinopathies [12][13][14][15]. Furthermore, the finding that different ␣SYN proteoforms could be detected in biological fluids (cerebrospinal fluid (CSF), plasma, red blood cells (RBC), and saliva [16][17][18][19][20][21][22][23][24][25][26][27][28][29] as well as in peripheral tissues (skin, intestinal mucosa, submandibular gland) [30][31][32][33] stimulated interest in ␣SYN as a potential disease biomarker for early diagnosis of PD, and for the differentiation between PD and other synucleinopathies [27,[34][35][36].…”

Comparative analysis of total alpha-synuclein (αSYN) immunoassays reveals that they do not capture the diversity of modified αSYN proteoforms

“…Less sensitive platforms might not be able to measure low-abundance species, therefore overlooking and de facto flattening the contribution of these proteins to total αSYN levels, compared to that obtained by ultrasensitive platforms with a much lower limit of detection. For instance, the increased αSYN levels in plasma from PD patients observed with the ultrasensitive Quanterix assay compared to other less sensitive methods could be related to the increased sensitivity of this platform, which may facilitate the detection and quantitation of low-abundance forms of αSYN, as discussed recently in [21]. On the other hand, assays with low dynamic ranges of their standard curves and low upper limits of quantification (ULOQ) will have decreased sensitivity and might account for saturation of the signal when certain species are highly abundant in the analyzed matrix.…”

“…This was mainly following the seminal discovery that mutations in the SNCA gene encoding αSYN cause familial PD [10,11] and that aggregated and posttranslationally modified (PTM) forms of αSYN, including phosphorylated, nitrated, ubiquitinated, and truncated species, are key components of Lewy bodies (LB) and Lewy neurites, the main neuropathological hallmark of PD and other synucleinopathies [12][13][14], [15]. Furthermore, the finding that different αSYN proteoforms could be detected in biological fluids (cerebrospinal fluid (CSF), plasma and red blood cells (RBC), and saliva) [16][17] [18][19][20][21][22][23][24][25][26][27][28][29] as well as in peripheral tissues (skin, intestinal mucosa, submandibular gland) [30][31][32][33] stimulated interest in αSYN as a potential disease biomarker for the early diagnosis of PD and for differentiation between PD and other synucleinopathies [25,[34][35][36].…”