Hyperefficient PrP Sc amplification of mouse-adapted BSE and scrapie strain by protein misfolding cyclic amplification technique.
Abnormal forms of prion protein (PrP(Sc)) accumulate via structural conversion of normal PrP (PrP(C)) in the progression of transmissible spongiform encephalopathy. Under cell-free conditions, the process can be efficiently replicated using in vitro PrP(Sc) amplification methods, including protein misfolding cyclic amplification. These methods enable ultrasensitive detection of PrP(Sc); however, there remain difficulties in utilizing them in practice. For example, to date, several rounds of protein misfolding cyclic amplification have been necessary to reach maximal sensitivity, which not only take several weeks, but also result in an increased risk of contamination. In this study, we sought to further promote the rate of PrP(Sc) amplification in the protein misfolding cyclic amplification technique using mouse transmissible spongiform encephalopathy models infected with either mouse-adapted bovine spongiform encephalopathy or mouse-adapted scrapie, Chandler strain. Here, we demonstrate that appropriate regulation of sonication dramatically accelerates PrP(Sc) amplification in both strains. In fact, we reached maximum sensitivity, allowing the ultrasensitive detection of < 1 LD(50) of PrP(Sc) in the diluted brain homogenates, after only one or two reaction rounds, and in addition, we detected PrP(Sc) in the plasma of mouse-adapted bovine spongiform encephalopathy-infected mice. We believe that these results will advance the establishment of a fast, ultrasensitive diagnostic test for transmissible spongiform encephalopathies.
Fujihara
Aiko
A
Department of Molecular Microbiology and Immunology, Nagasaki University Graduate School of Biomedical Sciences, Japan.
Atarashi
Ryuichiro
R
Fuse
Takayuki
T
Ubagai
Kaori
K
Nakagaki
Takehiro
T
Yamaguchi
Naohiro
N
Ishibashi
Daisuke
D
Katamine
Shigeru
S
Nishida
Noriyuki
N
D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D004072:Digitonin / Q000737:chemistry; D004492:Edetic Acid / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D016643:Encephalopathy, Bovine Spongiform / Q000378:metabolism*; D051379:Mice; D018620:PrPSc Proteins / Q000097:blood / Q000378:metabolism*; D017510:Protein Folding; D012608:Scrapie / Q000378:metabolism*
D018620:PrPSc Proteins; D004492:Edetic Acid; D004072:Digitonin