Background: The accumulation of aggregated α-synuclein (αSyn) is known as one of
the critical reasons to exhibit their variable molecular pathologies and phenotypes in synucleinopathies.
Recent studies suggested that the real-time quaking-induced conversion (RT-QuIC) assay
is one of the potential methods to detect these αSyn aggregates and could detect the aggregated
αSyn in the brain tissue and cerebrospinal fluid (CSF) using the propensity of the prion-like oligomerization.
Objective: We tried to optimize the αSyn RT-QuIC assay based on the aggregation of αSyn in
brain samples of synucleinopathies by comparing the conditions of the recently reported αSyn RTQuIC
Methods: This study applied a highly sensitive RT-QuIC assay using recombinant αSyn (rαSyn) to
detect aggregated αSyn in the brain tissue from dementia with Lewy bodies (DLB).
Results: This study compared αSyn RT-QuIC assays under conditions such as beads, rαSyn as a
substrate, reaction buffers, and fluorescence detectors. We observed that the addition of beads and
the use of 6x His-tagged rαSyn as a substrate help to obtain higher positive responses from αSyn
RT-QuIC assay seeding with brain homogenate (BH) of DLB and phosphate buffer-based reaction
showed higher positive responses than HEPES buffer-based reaction on both fluorescent microplate
readers. We also observed that the DLB BHs gave positive responses within 15–25h, which is
faster high positive responses than recently reported assays.
Conclusion: This established αSyn RT-QuIC assay will be able to apply to the early clinical diagnosis
of αSyn aggregates-related diseases in various biofluids such as CSF.