Objective: Optimizing of our sFIDA (surface-based fluorescence intensity distribution analysis) assay for specific detection and quantification of single α-synuclein aggregates.

Background: Parkinson’s disease (PD) is one of the most common neurodegenerative diseases characterized by loss of dopaminergic neurons in the brain and the formation of α-synuclein aggregates found in Lewy bodies throughout the brain. Diagnosis of PD is primarily based on clinical criteria. Therefore, identification of biomarkers would improve the diagnostic accuracy especially in early stages of parkinsonism. Owing to their fundamental role in PD pathology, α-synuclein aggregates are a promising biomarker for PD.

Methods: The sFIDA assay is able to detect and count single α-synuclein aggregates. The assay is based on a sandwich-ELISA biochemical setup combined with TIRF (total internal reflection fluorescence) microscopy-based readout. First, a monoclonal anti-α-synuclein capture antibody is covalently bound to the assay surface. After immobilizing of the target protein, aggregates are detected by adding two anti-α-synuclein antibody probes carrying two different fluorescent dyes. By choosing antibodies with overlapping epitopes for capturing and detection, surface-bound α-synuclein monomers cannot be recognized by the detection probes. In addition, only aggregates or oligomers are able to bind many detection antibodies and therefore show a very high local fluorescence signal.

Results: In order to assess the assay’s performance, we synthesized silica nanoparticles (SiNaPs) with conjugated recombinant α-synuclein as standard with a defined size of 20 nm. By spiking these α-synuclein SiNaPs in PBS, CSF and Plasma, respectively, we could detect these particles down to the femtomolar range in the sFIDA assay. In plasma environment, however, matrix effects lead to pronounced signal attenuation. First data on clinical CSF samples from a PD cohort will be presented.

Conclusions: sFIDA is a highly sensitive and specific assay for quantification of α-synuclein aggregates while it is insensitive towards monomers of α-synuclein. Applying specially designed standards allows us to derive quantitative information on the titer level of α-synuclein aggregates in human samples rendering sFIDA a promising tool for PD diagnostics.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/sfida-a-sensitive-diagnostic-assay-for-quantification-of-synuclein-aggregates/