Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Sensitive detection of pathological prion protein by cyclic amplification of protein misfolding

Abstract

Prions are the infectious agents responsible for transmissible spongiform encephalopathies. The principal component of prions is the glycoprotein PrPSc, which is a conformationally modified isoform of a normal cell-surface protein called PrPC (ref. 1). During the time between infection and the appearance of the clinical symptoms, minute amounts of PrPSc replicate by conversion of host PrPC, generating large amounts of PrPSc aggregates in the brains of diseased individuals. We aimed to reproduce this event in vitro. Here we report a procedure involving cyclic amplification of protein misfolding that allows a rapid conversion of large excess PrPC into a protease-resistant, PrPSc-like form in the presence of minute quantities of PrPSc template. In this procedure, conceptually analogous to polymerase chain reaction cycling, aggregates formed when PrPSc is incubated with PrPC are disrupted by sonication to generate multiple smaller units for the continued formation of new PrPSc. After cyclic amplification more than 97% of the protease-resistant PrP present in the sample corresponds to newly converted protein. The method could be applied to diagnose the presence of currently undetectable prion infectious agent in tissues and biological fluids, and may provide a unique opportunity to determine whether PrPSc replication results in the generation of infectivity in vitro.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Diagrammatic representation of the PMCA procedure.
Figure 2: Amplification of PrPSc by sonication cycles.
Figure 3: Sensitivity of the PMCA system.
Figure 4: Relationship between the extent of the conversion and the number of amplification cycles.

Similar content being viewed by others

References

  1. Prusiner, S. B. Prions. Proc. Natl Acad. Sci. USA 95, 13363–13383 (1998).

    Article  ADS  CAS  Google Scholar 

  2. Kocisko, D. A. et al. Cell-free formation of protease-resistant prion protein. Nature 370, 471–474 (1994).

    Article  ADS  CAS  Google Scholar 

  3. Horiuchi, M. & Caughey, B. Prion protein interconversions and the transmissible spongiform encephalopathies. Structure Fold Des. 7, R231–R240 (1999).

    Article  CAS  Google Scholar 

  4. Saborio, G. P. et al. Cell-lysate conversion of prion protein into its protease-resistant isoform suggests the participation of a cellular chaperone. Biochem. Biophys. Res. Commun. 258, 470–475 (1999).

    Article  CAS  Google Scholar 

  5. Telling, G. C. et al. Prion propagation in mice expressing human and chimeric PrP transgenes implicates the interaction of cellular PrP with another protein. Cell 83, 79–90 (1995).

    Article  CAS  Google Scholar 

  6. Aguzzi, A. & Weissmann, C. Prion research: the next frontier. Nature 389, 795–798 (1997).

    Article  ADS  CAS  Google Scholar 

  7. Cohen, F. E. & Prusiner, S. B. Pathologic conformations of prion proteins. Ann. Rev. Biochem. 67, 793–819 (1998).

    Article  CAS  Google Scholar 

  8. Caughey, B., Kocisko, D. A., Raymond, G. J. & Lansbury, P. T. Jr Aggregates of scrapie-associated prion protein induce the cell-free conversion of protease-sensitive prion protein to the protease-resistance state. Chem. Biol. 2, 807–817 (1995).

    Article  CAS  Google Scholar 

  9. Kocisko, D. A. et al. Species specificity in the cell-free conversion of prion protein to protease-resistant forms: a model for the scrapie species barrier. Proc. Natl Acad. Sci. USA 92, 3923–3927 (1995).

    Article  ADS  CAS  Google Scholar 

  10. Chabry, J., Caughey, B. & Chesebro, B. Specific inhibition of in vitro formation of protease-resistant prion protein by synthetic peptides. J. Biol. Chem. 273, 13203–13207 (1998).

    Article  CAS  Google Scholar 

  11. Caughey, W. S., Raymond, L. D., Horiuchi, M. & Caughey, B. Inhibition of protease-resistant prion protein formation by porphyrins and phthalocyanines. Proc. Natl Acad. Sci. USA 95, 12117–12122 (1998).

    Article  ADS  CAS  Google Scholar 

  12. Harper, J. D. & Lansbury, P. T. Jr Models of amyloid seeding in Alzheimer's disease and scrapie: mechanistic truths and physiological consequences of the time-dependent solubility of amyloid proteins. Ann. Rev. Biochem. 66, 385–407 (1997).

    Article  CAS  Google Scholar 

  13. Brown, P., Goldfarb, L. G. & Gajdusek, D. C. The new biology of spongiform encephalopathy: infectious amyloidoses with a genetic twist. Lancet 337, 1019–1022 (1991).

    Article  CAS  Google Scholar 

  14. Chesebro, B. BSE and prions: uncertainties about the agent. Science 279, 42–43 (1998).

    Article  ADS  CAS  Google Scholar 

  15. Mestel, R. Putting prions to the test. Science 273, 184–189 (1996).

    Article  ADS  CAS  Google Scholar 

  16. Dormont, D. Agents that cause transmissible subacute spongiform encephalopathies. Biomed. Pharmacother. 53, 3–8 (1999).

    Article  CAS  Google Scholar 

  17. Johnson, R. T. & Gibbs, C. J. Creutzfeldt–Jakob disease and related transmissible spongiform encephalopathies. New Engl. J. Med. 339, 1994–2004 (1998).

    Article  CAS  Google Scholar 

  18. Collinge, J. Variant Creutzfeldt–Jakob disease. Lancet 354, 317–323 (1999).

    Article  CAS  Google Scholar 

  19. Bruce, M. E. et al. Transmissions to mice indicate that new variant CJD is caused by the BSE agent. Nature 389, 498–501 (1997).

    Article  ADS  CAS  Google Scholar 

  20. Schiermeier, Q. Testing times for BSE. Nature 409, 658–659 (2001).

    Article  ADS  CAS  Google Scholar 

  21. Brown, P., Cervenakova, L. & Diringer, H. Blood infectivity and the prospects for a diagnostic screening test in Creutzfeldt–Jakob disease. J. Lab. Clin. Invest. 137, 5–13 (2001).

    CAS  Google Scholar 

  22. Glatzel, M. & Aguzzi, A. Peripheral pathogenesis of prion diseases. J. Gen. Virol. 81, 2813–2821 (2000).

    Article  CAS  Google Scholar 

  23. Kascsak, R. J. et al. Mouse polyclonal and monoclonal antibody to scrapie-associated fibril proteins. J. Virol. 61, 3688–3693 (1987).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank S. Peano, A. Conz, L. Anderes and M.-J. Frossard for technical assistance and R. J. Kascsak for providing 3F4 anti-PrP antibody. We are grateful to M. Pocchiari, S. Fumero, T. Wells, K. Maundrell and J. DeLamarter for reading the manuscript and providing comments. We also thank C. Herbert for help in the preparation of the figures.

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to Gabriela P. Saborio or Claudio Soto.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Saborio, G., Permanne, B. & Soto, C. Sensitive detection of pathological prion protein by cyclic amplification of protein misfolding. Nature 411, 810–813 (2001). https://doi.org/10.1038/35081095

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/35081095

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing