Amplification and Detection of Minuscule Amounts of Misfolded Prion Protein by Using the Real-Time Quaking-Induced Conversion

  • Matthias Schmitz
  • Niccolò Candelise
  • Franc Llorens
  • Inga Zerr
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1779)

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.

Key words

Cerebrospinal fluid Creutzfeldt-Jakob disease (CJD) Resistant prion protein Real-Time Quaking-Induced Conversion (RT-QuIC) 

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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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Matthias Schmitz
    • 1
  • Niccolò Candelise
    • 1
  • Franc Llorens
    • 2
  • Inga Zerr
    • 1
  1. 1.Department of NeurologyUniversity Medicine Goettingen and German Center for Neurodegenerative Diseases (DZNE) – site GöttingenGöttingenGermany
  2. 2.Center for Networked Biomedical Research on Neurodegenerative Diseases (CIBERNED)BarcelonaSpain

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