Optimization of a thermal shaker-aided quaking-induced conversion platform for the rapid detection of prion seeding activity in human samples
- 1Department of Immunological and Molecular Pharmacology, Faculty of Pharmaceutical Science, Fukuoka University, Fukuoka, Japan
Abstract
Creutzfeldt-Jakob disease (CJD) is a rapidly progressive, fatal neurodegenerative disorder caused by the misfolding of the normal prion protein (PrPC) into its pathogenic form (PrPSc). Therefore, effective clinical management and infection control require diagnostic assays that combine sub-attomole sensitivity with a turnaround time compatible with routine practice. While real-time quaking-induced conversion (RT-QuIC) offers excellent analytical performance, its protracted reaction time and dependence on plate readers that integrate high-speed shaking with continuous fluorescence detection restrict its widespread implementation.
Endpoint QuIC (EP-QuIC) is an established alternative approach that utilizes vigorous shaking in a ThermoMixer(R) C and relies on a single endpoint fluorescence readout. In this study, we introduced minor modifications to assay conditions to enhance the speed, robustness, and reproducibility of EP-QuIC. The refined assay detected prion seeding activity in sporadic CJD brain samples within 6 h--and, in some cases, as early as 2 h. Unlike RT-QuIC, problematic recombinant PrP batches that impaired assay reliability in RT-QuIC--exhibiting delayed kinetics and spontaneous ThT fluorescence in unseeded reactions--did not produce such effects in EP-QuIC. Instead, they yielded strong, specific fluorescence signals that clearly identified sCJD samples, demonstrating improved batch tolerance and diagnostic efficiency of this approach. When applied to CSF, the EP-QuIC assay showed 100% sensitivity and specificity, highlighting its potential as a rapid, reliable diagnostic tool for prion diseases.
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BibTeX
@misc{miyazaki2026optimization,
title = {{Optimization of a thermal shaker-aided quaking-induced conversion platform for the rapid detection of prion seeding activity in human samples}},
author = {Y. Miyazaki and T. Homma and T. Nakagaki and H. Takatsuki and T. Mori and R. Atarashi and K. Satoh and D. Ishibashi},
howpublished = {medRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.10.26362572},
url = {https://www.medrxiv.org/content/10.64898/2026.09.10.26362572v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - Optimization of a thermal shaker-aided quaking-induced conversion platform for the rapid detection of prion seeding activity in human samples
AU - Y. Miyazaki
AU - T. Homma
AU - T. Nakagaki
AU - H. Takatsuki
AU - T. Mori
AU - R. Atarashi
AU - K. Satoh
AU - D. Ishibashi
PY - 2026
DA - 2026/09/10
PB - medRxiv
DO - 10.64898/2026.09.10.26362572
UR - https://www.medrxiv.org/content/10.64898/2026.09.10.26362572v1
AB - Creutzfeldt-Jakob disease (CJD) is a rapidly progressive, fatal neurodegenerative disorder caused by the misfolding of the normal prion protein (PrPC) into its pathogenic form (PrPSc). Therefore, effective clinical management and infection control require diagnostic assays that combine sub-attomole sensitivity with a turnaround time compatible with routine practice. While real-time quaking-induced conversion (RT-QuIC) offers excellent analytical performance, its protracted reaction time and dependence on plate readers that integrate high-speed shaking with continuous fluorescence detection restrict its widespread implementation. Endpoint QuIC (EP-QuIC) is an established alternative approach that utilizes vigorous shaking in a ThermoMixer(R) C and relies on a single endpoint fluorescence readout. In this study, we introduced minor modifications to assay conditions to enhance the speed, robustness, and reproducibility of EP-QuIC. The refined assay detected prion seeding activity in sporadic CJD brain samples within 6 h--and, in some cases, as early as 2 h. Unlike RT-QuIC, problematic recombinant PrP batches that impaired assay reliability in RT-QuIC--exhibiting delayed kinetics and spontaneous ThT fluorescence in unseeded reactions--did not produce such effects in EP-QuIC. Instead, they yielded strong, specific fluorescence signals that clearly identified sCJD samples, demonstrating improved batch tolerance and diagnostic efficiency of this approach. When applied to CSF, the EP-QuIC assay showed 100% sensitivity and specificity, highlighting its potential as a rapid, reliable diagnostic tool for prion diseases.
ER -