期刊
NATURE METHODS
卷 4, 期 8, 页码 645-650出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/NMETH1066
关键词
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资金
- Intramural NIH HHS Funding Source: Medline
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 >= 50 ag of hamster PrPSc (approximate to 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-mu l 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.
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