Abstract
A characteristic feature of transmissible spongiform encephalopathies (TSE) is the progressive accumulation of protein aggregates in the brain in a self-propagation manner. Based on this mechanism, in vitro protein amplification systems (such as real-time quaking-induced conversion (RT-QuIC)) for the detection of misfolded prion protein scrapie (PrPres) in CSF were a major step in pre-mortem diagnosis of human prion diseases. Here, we describe a protocol of the RT-QuIC assay to detect PrPres in CSF of prion disease patients. This methodology depends on prion seeds that induce misfolding and aggregation of a substrate by cycles of incubation and quaking. Besides diagnostics, further applications of the RT-QuIC appear to be promising for discrimination between different PrP subtypes or strains, understanding the mechanism of protein misfolding and pre-screening of anti-prion drugs. The technique can be further developed to be used to study characteristics of misfolded proteins in other “prion like” diseases, such as tauopathies, synucleinopathies, or amyloidopathies.
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Abbreviations
- AUC:
-
Relative area under the curve
- CJD:
-
Creutzfeldt-Jakob disease
- CSF:
-
Cerebrospinal fluid
- PK:
-
Proteinase K
- PrPC:
-
Cellular prion protein
- PrPres:
-
Resistant prion protein
- rcf:
-
Relative centrifugal force
- recPrP:
-
Recombinant PrP
- rfu:
-
Relative fluorescence units
- rpm:
-
Rounds per minute
- RT:
-
Room temperature
- RT-QuIC:
-
Real-time quaking-induced conversion
- s:
-
Second
- Th-T:
-
Thioflavin T
- TSE:
-
Transmissible spongiform encephalopathies
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Schmitz, M., Candelise, N., Llorens, F., Zerr, I. (2018). Amplification and Detection of Minuscule Amounts of Misfolded Prion Protein by Using the Real-Time Quaking-Induced Conversion. In: Sigurdsson, E., Calero, M., Gasset, M. (eds) Amyloid Proteins. Methods in Molecular Biology, vol 1779. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7816-8_16
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DOI: https://doi.org/10.1007/978-1-4939-7816-8_16
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