Establishment Of Method For The Determination Of Aggregated Alpha-Synuclein In Dlb Patient Using Rt-Quic Assay

Background: The accumulation of aggregated alpha-synuclein (alpha Syn) 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 alpha Syn aggregates and could detect the aggregated alpha Syn in the brain tissue and cerebrospinal fluid (CSF) using the propensity of the prion-like oligomerization.Objective: We tried to optimize the alpha Syn RT-QuIC assay based on the aggregation of alpha Syn in brain samples of synucleinopathies by comparing the conditions of the recently reported alpha Syn RT-QuIC assays.Methods: This study applied a highly sensitive RT-QuIC assay using recombinant alpha Syn (r alpha Syn) to detect aggregated alpha Syn in the brain tissue from dementia with Lewy bodies (DLB).Results: This study compared alpha Syn RT-QuIC assays under conditions such as beads, r alpha Syn as a substrate, reaction buffers, and fluorescence detectors. We observed that the addition of beads and the use of 6x His-tagged r alpha Syn as a substrate help to obtain higher positive responses from alpha Syn RT-QuIC assay seeding with brain homogenate (BH) of DLB and phosphate buffer-based reaction showed higher positive responses than FIEPES buffer-based reaction on both fluorescent micro-plate 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 alpha Syn RT-QuIC assay will be able to apply to the early clinical diagnosis of alpha Syn aggregates-related diseases in various biofluids such as CSF.