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
Part of the Methods in Molecular Biology book series (MIMB, volume 1779)


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) 



Relative area under the curve


Creutzfeldt-Jakob disease


Cerebrospinal fluid


Proteinase K


Cellular prion protein


Resistant prion protein


Relative centrifugal force


Recombinant PrP


Relative fluorescence units


Rounds per minute


Room temperature


Real-time quaking-induced conversion




Thioflavin T


Transmissible spongiform encephalopathies


  1. 1.
    Prusiner SB, Kingsbury DT (1985) Prions--infectious pathogens causing the spongiform encephalopathies. CRC Crit Rev Clin Neurobiol 1:181–200PubMedGoogle Scholar
  2. 2.
    Pan KM, Baldwin M, Nguyen J et al (1993) Conversion of alpha-helices into beta-sheets features in the formation of the scrapie prion proteins. Proc Natl Acad Sci U S A 90:10962–10966CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Saborio GP, Permanne B, Soto C (2001) Sensitive detection of pathological prion protein by cyclic amplification of protein misfolding. Nature 441:810–813CrossRefGoogle Scholar
  4. 4.
    Morales R, Duran-Aniotz C, Diaz-Espinosa R et al (2014) Protein misfolding cyclic amplification of infectious prions. Nat Protoc 7:1397–1409CrossRefGoogle Scholar
  5. 5.
    Atarashi R, Satoh K, Sano K et al (2011) Ultrasensitive human prion detection in cerebrospinal fluid by real time quaking-induced conversion. Nat Med 17:175–178CrossRefPubMedGoogle Scholar
  6. 6.
    Khurana R, Coleman C, Ionescu-Zanetti C et al (2005) Mechanism of thioflavin T binding to amyloid fibrils. J Struct Biol 151:229–238CrossRefPubMedGoogle Scholar
  7. 7.
    McGuire LI, Peden AH, Orrú CD et al (2012) Real time quaking-induced conversion analysis of cerebrospinal fluid in sporadic Creutzfeldt-Jakob disease. Ann Neurol 72:278–285CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Cramm M, Schmitz M, Karch A et al (2016) Stability and reproducibility underscore utility of RT-QuIC for diagnosis of Creutzfeldt-Jakob disease. Mol Neurobiol 53:1896–1904CrossRefPubMedGoogle Scholar
  9. 9.
    Cramm M, Schmitz M, Karch A et al (2015) Characteristic CSF-prion seeding activity in humans with prion diseases. Mol Neurobiol 51:396–405CrossRefPubMedGoogle Scholar
  10. 10.
    Schmitz M, Cramm M, Llorens F et al (2016) Application of an in vitro-amplification assay as a novel pre-screening test for compounds inhibiting the aggregation of prion protein scrapie. Sci Rep 6:28711CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Fairfoul G, McGuire LI, Pal S et al (2016) α-synuclein RT-QuIC in the CSF of patients with α-synucleinopathies. Ann Clin Transl Neurol 3:812–818CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Salvadores N, Shahnawaz M, Scarpini E et al (2014) Detection of misfolded Abeta oligomers for sensitive biochemical diagnosis of Alzheimer’s disease. Cell Rep 10:261–268CrossRefGoogle Scholar
  13. 13.
    Meyer V, Dinkel PD, Rickman Hager E et al (2014) Amplification of tau fibrils from minute quantities of seeds. Biochemistry 53:5804–5809CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Saijo E, Ghetti B, Zanusso G et al (2017) Ultrasensitive and selective detection of 3-repeat tau seeding activity in Pick disease brain and cerebrospinal fluid. Acta Neuropathol 133:751–765CrossRefPubMedGoogle Scholar
  15. 15.
    Schmitz M, Cramm M, Llorens F et al (2016) Adapting the real-time quaking-induced conversion assay to multiple applications in human prion disease management. Nat Protoc 11:2233–2242CrossRefPubMedGoogle Scholar
  16. 16.
    Orrú CD, Wilham JM, Raymond LD et al (2011) Prion disease blood test using immunoprecipitation and improved quaking induced conversion. MBio 2:e00078–e00011CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Wilham JM, Wilham JM, Orrú CD et al (2010) Rapid end-point quantitation of prion seeding activity with sensitivity comparable to bioassays. PLoS Pathog 6:e1001217CrossRefPubMedPubMedCentralGoogle Scholar

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

Personalised recommendations