Objective: Early diagnosis for PD is disturbed by the lack of sensitive and objective detection of accumulated αSyn in the patient’s brain. Recently, Real-Time Quaking-Induced Conversion (RT-QuIC) analysis has been developed for a sensitive and specific detection of misfolded synuclein in the CSF of PD patients. Using these methods, we developed a new devise “HANABI (HANdai Amyloid Burst Inducer)” to induce the amplification of misfolded αSyn using ultrasonication automatically. HANABI dramatically reduced the detection time compare to the former methods.

Background: The RT-QuIC technique is based on amyloid seed–induced misfolding and aggregation of recombinant protein substrate, accelerated by alternating cycles of shaking and rest in fluorescence plate readers. Recent studies showed RT-QuIC analysis were also able to detect theαSyn amyloid in the CSF of PD patients. Although the CSF RT‐QuIC has been shown to be an accurate diagnostic test for PD, with high sensitivity (95%) and specificity (100%), the protocol takes ~100 hr to complete. Using these techniques, we established a new device ‘HANABI’ to induce the amplification ofαSyn amyloid, which enable to shorten the assay duration for several hours.

Methods:  The RT-QuiC reaction buffer (RB) was composed of 150 mmol/L NaCl, 50mM Tris HCl pH7.4, 10 lmol/L thio- flavin T (ThT), and 1.4 mg/mL human recombinant full-length (1–140 aa) αSyn. Each well of a black 96-well plate contained 90ul RB. Reactions were seeded with 10 ul of CSF. The plates were incubated in a HANABI, with which the ultrasonication-forced fibrillation of proteins can be automatically and rapidly analyzed, at 37°C for 6 h with intermittent sonication cycles: 3 min sonication, 7 min rest. ThT fluorescence measurements (450 nm excitation and 480 nm emission) were taken every 10 min.

Results: We employed 10 PD patients and 7 disease control patients. This new assay could detectαSyn aggregation in PD cerebrospinal fluid with sensitivities of 80% and with specificity of 70% when compared to control cerebrospinal fluid. From sample preparation to analysis of results, the protocol takes only ~7h to complete.

Conclusions: These results suggest that our new analysis of cerebrospinal fluid is potentially useful for the early clinical assessment of patients with α- synucleinopathy

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MDS Abstracts - https://www.mdsabstracts.org/abstract/new-device-hanabi-handai-amyloid-burst-inducer-is-a-rapid-and-sensitive-detecting-system-of-%ce%b1-synuclein-fibril-in-csf-from-parkinsons-disease-patients/