Prion detection by an amyloid seeding assay

*Institute for Neurodegenerative Diseases and
^¶Departments of Neurology and Biochemistry and Biophysics, University of California, San Francisco, CA 94143; and
^§Institut für Physikalische Biologie, Heinrich-Heine Universität, 40225 Düsseldorf, Germany

Contributed by Stanley B. Prusiner, October 25, 2007 (received for review September 18, 2007)

Abstract

Polymerization of recombinant prion protein (recPrP), which was produced in bacteria, into amyloid fibers was accompanied by the acquisition of prion infectivity. We report here that partially purified preparations of prions seed the polymerization of recPrP into amyloid as detected by a fluorescence shift in the dye Thioflavin T. Our amyloid seeding assay (ASA) detected PrP^Sc, the sole component of the prion, in brain samples from humans with sporadic Creutzfeldt–Jakob disease, as well as in rodents with experimental prion disease. The ASA detected a variety of prion strains passaged in both mice and hamsters. The sensitivity of the ASA varied with strain type; for hamster Sc237 prions, the limit of detection was ≈1 fg. Some prion strains consist largely of protease-sensitive PrP^Sc (sPrP^Sc), and these strains were readily detected by ASA. Our studies show that the ASA provides an alternative methodology for detecting both sPrP^Sc and protease-resistant PrP^Sc that does not rely on protease digestion or immunodetection.

Footnotes

^‖To whom correspondence should be addressed. E-mail: stanley{at}ind.ucsf.edu
Author contributions: D.W.C., Q.Z., G.L., D.R., and S.B.P. designed research; D.W.C., Q.Z., S.W., and D.G. performed research; D.W.C. and S.B.P. analyzed data; and D.W.C. and S.B.P. wrote the paper.
↵ ^†Present address: Buck Institute for Age Research, Novato, CA 94945.
↵ ^‡Present address: Neurobiology Sector, Scuola Internazionale Superiore di Studi Avanzati, Trieste 34012, Italy.
Conflict of interest statement: D.G., G.L., D.R., and S.B.P. have financial interest in InPro Biotechnology, Inc.
This article contains supporting information online at www.pnas.org/cgi/content/full/0710152105/DC1.