Validation of a quantitative cerebrospinal fluid alpha-synuclein assay in a European-wide interlaboratory study

Kruse, Niels; Persson, Staffan; Alcolea, Daniel; Bahl, Justyna M.C.; Baldeiras, Inês; Capello, Elisabetta; Chiasserini, Davide; Bocchio-Chiavetto, Luisella; Emeršič, Andreja; Engelborghs, Sebastiaan; Eren, Erden; Fladby, Tormod; Frisoni, Giovanni B.; García‐Ayllón, María‐Salud; Genç, Şermin; Gkatzima, Olymbia; Heegaard, Niels H. H.; Janeiro, André M.; Kováčech, Branislav; Kuiperij, H. Bea; Leitão, Maria João; Lleó, Alberto; Martins, Madalena; Matos, Maria Margarida Nunes Gaspar de; Møllergård, Hanne M.; Nobili, Flavio; Öhrfelt, Annika; Parnetti, Lucilla; Oliveira, Catarina R.; Rot, Uroš; Sáez‐Valero, Javier; Struyfs, Hanne; Tanassi, Julia T.; Taylor, Peggy; Tsolaki, Magda; Vanmechelen, Eugeen; Verbeek, Marcel M.; Žilka, Norbert; Blennow, Kaj; Zetterberg, Henrik; Mollenhauer, Brit

doi:10.1016/j.neurobiolaging.2015.05.003

Cited by 34 publications

(37 citation statements)

References 27 publications

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“…Several asyn specific assays have been developed to measure levels of total asyn protein and its modified variants in brain tissue, CSF and plasma samples with the goal to find a wet biomarker for PD and other synucleinopathies [17, 18, 21, 27, 28]. As pS129 asyn comprises a large part of asyn found in inclusions [14], it has been of interest to further measure pS129 asyn levels in various samples collected from patients.…”

Section: Discussionmentioning

confidence: 99%

A novel multiplex assay for simultaneous quantification of total and S129 phosphorylated human alpha-synuclein

Landeck¹,

Hall²,

Ardah³

et al. 2016

Mol Neurodegeneration

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BackgroundAlpha-synuclein (asyn) has been shown to play an important role in the neuropathology of Parkinson’s disease (PD). In the diseased brain, classic intraneuronal inclusions called Lewy bodies contain abnormal formations of asyn protein which is mostly phosphorylated at serine 129 (pS129 asyn). This suggests that post-translational modifications may play a role in the pathogenic process. To date, several uniplex assays have been developed in order to quantify asyn not only in the brain but also in cerebrospinal fluid and blood samples in order to correlate asyn levels to disease severity and progression. Notably, only four assays have been established to measure pS129 asyn specifically and none provide simultaneous readout of the total and pS129 species. Therefore, we developed a sensitive high-throughput duplex assay quantifying total and pS129 human asyn (h-asyn) in the same well hence improving accuracy as well as saving time, consumables and samples.ResultsUsing our newly established duplex assay we measured total and pS129 h-asyn in vitro showing that polo-like kinase 2 (PLK2) can phosphorylate asyn up to 41 % in HEK293 cells and in vivo the same kinase phosphorylated h-asyn up to 17 % in rat ventral midbrain neurons. Interestingly, no increase in phosphorylation was observed when PLK2 and h-asyn were co-expressed in rat striatal neurons. Furthermore, using this assay we investigated h-asyn levels in brain tissue samples from patients with PD as well as PD dementia and found significant differences in pS129 h-asyn levels not only between disease tissue and healthy control samples but also between the two distinct disease states especially in hippocampal tissue samples.ConclusionsThese results demonstrate that our duplex assay for simultaneous quantification is a useful tool to study h-asyn phosphorylation events in biospecimens and will be helpful in studies investigating the precise causative link between post-translational modification of h-asyn and PD pathology.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-016-0125-0) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 99%

A novel multiplex assay for simultaneous quantification of total and S129 phosphorylated human alpha-synuclein

Landeck¹,

Hall²,

Ardah³

et al. 2016

Mol Neurodegeneration

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“…These platforms depend on antibodies, leading to variability in their inherent efficiency, as well as substantial differences between assays using different antibody pairs, complications due to matrix effects in samples, and difficulty in measuring the absolute concentration of the markers. The standardization of CSF tau and Aβ 1–42 measurements using immunoassays between laboratories has proven difficult (15), (16), and this phenomenon is not unique to the AD field (17). For example, a mass spectrometry-based assay has been developed to quantify synuclein isoforms in CSF, showing the value of measuring more than one marker to increase the potential of the assay (18).…”

Section: Introductionmentioning

confidence: 99%

Mass-Spectrometry-Based Method To Quantify in Parallel Tau and Amyloid β 1–42 in CSF for the Diagnosis of Alzheimer’s Disease

Pottiez

Stewart

et al. 2017

J. Proteome Res.

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Alzheimer’s disease (AD), the most common form of dementia, afflicts about 50 million people worldwide. Currently, AD diagnosis is primarily based on psychological evaluation and can only be confirmed post-mortem. Reliable and objective biomarkers for prognosis and diagnosis have been sought for several years. Together, tau and amyloid β 1–42 (Aβ42) in cerebrospinal fluid (CSF) have been shown to provide good diagnostic sensitivity and specificity. Additionally, phosphorylated forms of tau, such as tau pS181, have also shown promising results. However, the measurement of such markers currently relies on antibody-based immunoassays that have shown variability, leading to discrepant results across laboratories. To date, mass spectrometry methods developed to evaluate CSF tau and Aβ42 are not compatible. We present in this article the development of a mass spectrometry-based method of quantification for CSF tau and Aβ42 in parallel. The absolute concentration of tau and Aβ42 we measured are on average 50 ng/mL (7–130 ng/mL) and 7.1 ng/mL (3–13 ng/mL), respectively. Analyses of CSF tau and Aβ42, in a cohort of patients with AD, mild cognitive impairment and healthy controls (30 subjects), provide significant group differences evaluated with ROC curves (AUC(control-AD) and AUC(Control-MCI)=1, AUC(MCI-AD)=0.76), with at least equivalent diagnostic utility to immunoassay measurements in the same sample set. Finally, a significant and negative correlation was found between the tau and Aβ peptide ratio and the disease severity.

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“…The higher site and total variability with the BioLegend assay is driven by measurements from one laboratory using a plate reader that was off in calibration (Kruse et al . ). If that laboratory is removed, BioLegend's variance components return to levels commensurate with the other assays (inter‐laboratory CV 1.3–20.5%).…”

Section: Resultsmentioning

confidence: 97%

“…The assays showed excellent within‐laboratory performance and round robin results showed that three out of four assays (Roche, ADx, and MSD) gave almost identical results in independent laboratories, whereas the BioLegend ELISA showed some inter‐laboratory variability, mostly driven by the results from one laboratory and likely attributable to variability with ELISA plate reader calibration, which has been observed with this assay during a multi‐laboratory comparison study in the past (Kruse et al . ).…”

Section: Discussionmentioning

confidence: 97%

Antibody‐based methods for the measurement of α‐synuclein concentration in human cerebrospinal fluid – method comparison and round robin study

Mollenhauer

Bowman

Drake

et al. 2018

Journal of Neurochemistry

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α-Synuclein is the major component of Lewy bodies and a candidate biomarker for neurodegenerative diseases in which Lewy bodies are common, including Parkinson's disease and dementia with Lewy bodies. A large body of literature suggests that these disorders are characterized by reduced concentrations of α-synuclein in cerebrospinal fluid (CSF), with overlapping concentrations compared to healthy controls and variability across studies. Several reasons can account for this variability, including technical ones, such as inter-assay and inter-laboratory variation (reproducibility). We compared four immunochemical methods for the quantification of α-synuclein concentration in 50 unique CSF samples. All methods were designed to capture most of the existing α-synuclein forms in CSF ('total' α-synuclein). Each of the four methods showed high analytical precision, excellent correlation between laboratories (R 0.83-0.99), and good correlation with each other (R 0.64-0.93), although the slopes of the regression lines were different between the four immunoassays. The use of common reference CSF samples decreased the differences in α-synuclein concentration between detection methods and technologies. Pilot data on an immunoprecipitation mass spectrometry (IP-MS) method is also presented. Our results suggest that the four immunochemical methods and the IP-MS method measure similar forms of α-synuclein and that a common reference material would allow harmonization of results between immunoassays.

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Validation of a quantitative cerebrospinal fluid alpha-synuclein assay in a European-wide interlaboratory study

Cited by 34 publications

References 27 publications

A novel multiplex assay for simultaneous quantification of total and S129 phosphorylated human alpha-synuclein

A novel multiplex assay for simultaneous quantification of total and S129 phosphorylated human alpha-synuclein

Mass-Spectrometry-Based Method To Quantify in Parallel Tau and Amyloid β 1–42 in CSF for the Diagnosis of Alzheimer’s Disease

Antibody‐based methods for the measurement of α‐synuclein concentration in human cerebrospinal fluid – method comparison and round robin study

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