Patients with isolated REM-sleep behavior disorder have elevated levels of alpha-synuclein aggregates in stool.

Misfolded and aggregated α-synuclein is a neuropathological hallmark of Parkinson's disease (PD). Thus, α-synuclein aggregates are regarded as a biomarker for the development of diagnostic assays. Quantification of α-synuclein aggregates in body fluids is challenging, and requires highly sensitive and specific assays. Recent studies suggest that α-synuclein aggregates may be shed into stool. We used surface-based fluorescence intensity distribution analysis (sFIDA) to detect and quantify single particles of α-synuclein aggregates in stool of 94 PD patients, 72 isolated rapid eye movement sleep behavior disorder (iRBD) patients, and 51 healthy controls. We measured significantly elevated concentrations of α-synuclein aggregates in stool of iRBD patients versus those of controls (p = 0.024) or PD patients (p < 0.001). Our results show that α-synuclein aggregates are excreted in stool and can be measured using the sFIDA assay, which could support the diagnosis of prodromal synucleinopathies.

Quantitative detection of α-Synuclein and Tau oligomers and other aggregates by digital single particle counting.

The pathological hallmark of neurodegenerative diseases is the formation of toxic oligomers by proteins such as alpha-synuclein (aSyn) or microtubule-associated protein tau (Tau). Consequently, such oligomers are promising biomarker candidates for diagnostics as well as drug development. However, measuring oligomers and other aggregates in human biofluids is still challenging as extreme sensitivity and specificity are required. We previously developed surface-based fluorescence intensity distribution analysis (sFIDA) featuring single-particle sensitivity and absolute specificity for aggregates. In this work, we measured aSyn and Tau aggregate concentrations of 237 cerebrospinal fluid (CSF) samples from five cohorts: Parkinson's disease (PD), dementia with Lewy bodies (DLB), Alzheimer's disease (AD), progressive supranuclear palsy (PSP), and a neurologically-normal control group. aSyn aggregate concentration discriminates PD and DLB patients from normal controls (sensitivity 73%, specificity 65%, area under the receiver operating curve (AUC) 0.68). Tau aggregates were significantly elevated in PSP patients compared to all other groups (sensitivity 87%, specificity 70%, AUC 0.76). Further, we found a tight correlation between aSyn and Tau aggregate titers among all patient cohorts (Pearson coefficient of correlation r = 0.81). Our results demonstrate that aSyn and Tau aggregate concentrations measured by sFIDA differentiate neurodegenerative disease diagnostic groups. Moreover, sFIDA-based Tau aggregate measurements might be particularly useful in distinguishing PSP from other parkinsonisms. Finally, our findings suggest that sFIDA can improve pre-clinical and clinical studies by identifying those individuals that will most likely respond to compounds designed to eliminate specific oligomers or to prevent their formation.

Nanoparticle standards for immuno-based quantitation of α-synuclein oligomers in diagnostics of Parkinson's disease and other synucleinopathies.

Parkinson's disease (PD) is a neurodegenerative disorder that is characterized by symptoms such as rigor, tremor and bradykinesia. A reliable and early diagnosis could improve the development of early therapeutic strategies before death of dopaminergic neurons leads to the first clinical symptoms. The sFIDA (surface-based fluorescence intensity distribution analysis) assay is a highly sensitive method to determine the concentration of α-synuclein (α-syn) oligomers which are presumably the major toxic isoform of α-syn and potentially the most direct biomarker for PD. Oligomer-based diagnostic tests require standard molecules that closely mimic the native oligomer. This is particularly important for calibration and assessment of inter-assay variation. In this study, we generated a standard in form of α-syn coated silica nanoparticles (α-syn-SiNaPs) that are in the size range of α-syn oligomers and provide a defined number of α-syn epitopes. The preparation of the sFIDA assay was realized on an automated platform to allow handling of high number of samples and reduce the effects of human error. The assay outcome was analyzed by determination of coefficient of variation and linearity for the applied α-syn-SiNaPs concentrations. Additionally, the limit of detection and lower limit of quantification were determined yielding concentrations in the lower femtomolar range.

sFIDA automation yields sub-femtomolar limit of detection for Aß aggregates in body fluids.

OBJECTIVES: Alzheimer's disease (AD) is a neurodegenerative disorder with yet non-existent therapeutic and limited diagnostic options. Reliable biomarker-based AD diagnostics are of utmost importance for the development and application of therapeutic substances. We have previously introduced a platform technology designated 'sFIDA' for the quantitation of amyloid ß peptide (Aß) aggregates as AD biomarker. In this study we implemented the sFIDA assay on an automated platform to enhance robustness and performance of the assay. DESIGN AND METHODS: In sFIDA (surface-based fluorescence intensity distribution analysis) Aß species are immobilized by a capture antibody to a glass surface. Aß aggregates are then multiply loaded with fluorescent antibodies and quantitated by high resolution fluorescence microscopy. As a model system for Aß aggregates, we used Aß-conjugated silica nanoparticles (Aß-SiNaPs) diluted in PBS buffer and cerebrospinal fluid, respectively. Automation of the assay was realized on a liquid handling system in combination with a microplate washer. RESULTS: The automation of the sFIDA assay results in improved intra-assay precision, linearity and sensitivity in comparison to the manual application, and achieved a limit of detection in the sub-femtomolar range. CONCLUSIONS: Automation improves the precision and sensitivity of the sFIDA assay, which is a prerequisite for high-throughput measurements and future application of the technology in routine AD diagnostics.

Analysis of anticoagulants for blood-based quantitation of amyloid ß oligomers in the sFIDA assay.

Early diagnostics at the preclinical stage of Alzheimer's disease is of utmost importance for drug development in clinical trials and prognostic guidance. Since soluble Aß oligomers are considered to play a crucial role in the disease pathogenesis, several methods aim to quantify Aß oligomers in body fluids such as cerebrospinal fluid (CSF) and blood plasma. The highly specific and sensitive method surface-based fluorescence intensity distribution analysis (sFIDA) has successfully been established for oligomer quantitation in CSF samples. In our study, we explored the sFIDA method for quantitative measurements of synthetic Aß particles in blood plasma. For this purpose, EDTA-, citrate- and heparin-treated blood plasma samples from five individual donors were spiked with Aß coated silica nanoparticles (Aß-SiNaPs) and were applied to the sFIDA assay. Based on the assay parameters linearity, coefficient of variation and limit of detection, we found that EDTA plasma yields the most suitable parameter values for quantitation of Aß oligomers in sFIDA assay with a limit of detection of 16 fM.

Biofunctionalized Silica Nanoparticles: Standards in Amyloid-ß Oligomer-Based Diagnosis of Alzheimer's Disease.

Amyloid-ß (Aß) oligomers represent a promising biomarker for the early diagnosis of Alzheimer's disease (AD). However, state-of-the-art methods for immunodetection of Aß oligomers in body fluids show a large variability and lack a reliable and stable standard that enables the reproducible quantitation of Aß oligomers. At present, the only available standard applied in these assays is based on a random aggregation process of synthetic Aß and has neither a defined size nor a known number of epitopes. In this report, we generated a highly stable standard in the size range of native Aß oligomers that exposes a defined number of epitopes. The standard consists of a silica nanoparticle (SiNaP), which is functionalized with Aß peptides on its surface (Aß-SiNaP). The different steps of Aß-SiNaP synthesis were followed by microscopic, spectroscopic and biochemical analyses. To investigate the performance of Aß-SiNaPs as an appropriate standard in Aß oligomer immunodetection, Aß-SiNaPs were diluted in cerebrospinal fluid and quantified down to a concentration of 10 fM in the sFIDA (surface-based fluorescence intensity distribution analysis) assay. This detection limit corresponds to an Aß concentration of 1.9 ng l-1 and lies in the sensitivity range of currently applied diagnostic tools based on Aß oligomer quantitation. Thus, we developed a highly stable and well-characterized standard for the application in Aß oligomer immunodetection assays that finally allows the reproducible quantitation of Aß oligomers down to single molecule level and provides a fundamental improvement for the worldwide standardization process of diagnostic methods in AD research.

Application of an Amyloid Beta Oligomer Standard in the sFIDA Assay.

Still, there is need for significant improvements in reliable and accurate diagnosis for Alzheimer's disease (AD) at early stages. It is widely accepted that changes in the concentration and conformation of amyloid-ß (Aß) appear several years before the onset of first symptoms of cognitive impairment in AD patients. Because Aß oligomers are possibly the major toxic species in AD, they are a promising biomarker candidate for the early diagnosis of the disease. To date, a variety of oligomer-specific assays have been developed, many of them ELISAs. Here, we demonstrate the sFIDA assay, a technology highly specific for Aß oligomers developed toward single particle sensitivity. By spiking stabilized Aß oligomers to buffer and to body fluids from control donors, we show that the sFIDA readout correlates with the applied concentration of stabilized oligomers diluted in buffer, cerebrospinal fluid (CSF), and blood plasma over several orders of magnitude. The lower limit of detection was calculated to be 22 fM of stabilized oligomers diluted in PBS, 18 fM in CSF, and 14 fM in blood plasma.