Ultrasensitive detection of scrapie prion protein using seeded conversion of recombinant prion protein.
The scrapie prion protein isoform, PrPSc, is a prion-associated marker that seeds the conformational conversion and polymerization of normal protease-sensitive prion protein (PrP-sen). This seeding activity allows ultrasensitive detection of PrPSc using cyclical sonicated amplification (PMCA) reactions and brain homogenate as a source of PrP-sen. Here we describe a much faster seeded polymerization method (rPrP-PMCA) which detects >or=50 ag of hamster PrPSc (approximately 0.003 lethal dose) within 2-3 d. This technique uses recombinant hamster PrP-sen, which, unlike brain-derived PrP-sen, can be easily concentrated, mutated and synthetically tagged. We generated protease-resistant recombinant PrP fibrils that differed from spontaneously initiated fibrils in their proteolytic susceptibility and by their infrared spectra. This assay could discriminate between scrapie-infected and uninfected hamsters using 2-microl aliquots of cerebral spinal fluid. This method should facilitate the development of rapid, ultrasensitive prion assays and diagnostic tests, in addition to aiding fundamental studies of structure and mechanism of PrPSc formation.
Atarashi
Ryuichiro
R
Laboratory of Persistent Viral Diseases, National Institutes of Health, 903 S. 4th Street, Hamilton, Montana 59840, USA.
Moore
Roger A
RA
Sim
Valerie L
VL
Hughson
Andrew G
AG
Dorward
David W
DW
Onwubiko
Henry A
HA
Priola
Suzette A
SA
Caughey
Byron
B
D000818:Animals; D015153:Blotting, Western; D002555:Cerebrospinal Fluid; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D051379:Mice; D008854:Microscopy, Electron; D018620:PrPSc Proteins / Q000032:analysis*; D011994:Recombinant Proteins / Q000032:analysis; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet
D018620:PrPSc Proteins; D011994:Recombinant Proteins