Prion detection by an amyloid seeding assay.
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 approximately 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.
Colby
David W
DW
Institute for Neurodegenerative Diseases [corrected], University of California, San Francisco, CA 94143, USA.
Zhang
Qiang
Q
Wang
Shuyi
S
Groth
Darlene
D
Legname
Giuseppe
G
Riesner
Detlev
D
Prusiner
Stanley B
SB
D000682:Amyloid / Q000737:chemistry; D000818:Animals; D052160:Benzothiazoles; D001921:Brain / Q000378:metabolism; D007562:Creutzfeldt-Jakob Syndrome / Q000175:diagnosis / Q000378:metabolism; D006224:Cricetinae; D006801:Humans; D007700:Kinetics; D008647:Mesocricetus; D051379:Mice; D010447:Peptide Hydrolases / Q000378:metabolism; D011108:Polymers / Q000378:metabolism; D011328:Prions / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods; D013844:Thiazoles / Q000737:chemistry
D000682:Amyloid; D052160:Benzothiazoles; D011108:Polymers; D011328:Prions; D011994:Recombinant Proteins; D013844:Thiazoles; C009462:thioflavin T; D010447:Peptide Hydrolases