sFIDA: A Sensitive Diagnostic Assay For α-Synuclein Aggregates

Abstract

ObjectiveOptimizing of our sFIDA (surface-based fluorescence intensity distribution analysis) assay which is able to specifically detect and quantitate α-synuclein aggregates. BackgroundParkinson disease (PD) leads to motoric disfunctions and 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 thoughout the brain. Diagnosis of PD is primarily based on clinical criteria. Therefore, identification of suitable biomarkers would facilitate the differential diagnosis in patients with early stages of parkinsonism.MethodssFIDA 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, an antibody is covalently bound to a glass surface. After binding of the target protein to the capture antibody, fluorescence-labeled detection antibodies are added, and the surface is scanned by laser beams. Protein aggregates are able to bind many detection antibodies and therefore lead to a very high local fluorescence signal. ResultsThe assay was optimized by testing several assay conditions and different α-synuclein antibodies. Synthetic α-synuclein aggregates in cerebrospinal fluid (CSF) were easily detected down to the femtomolar range. ConclusionssFIDA is a highly sensitive and specific assay to specifically quantitate α-synuclein aggregates and to distinguish between aggregates and monomers of α-synuclein