title: Rapid and Quantitative Assay of Amyloid-Seeding Activity in Human Brains Affected with Prion Diseases.
abstract: The infectious agents of the transmissible spongiform encephalopathies are composed of amyloidogenic prion protein, PrPSc. Real-time quaking-induced conversion can amplify very small amounts of PrPSc seeds in tissues/body fluids of patients or animals. Using this in vitro PrP-amyloid amplification assay, we quantitated the seeding activity of affected human brains. End-point assay using serially diluted brain homogenates of sporadic Creutzfeldt-Jakob disease patients demonstrated that 50% seeding dose (SD50) is reached approximately 10(10)/g brain (values varies 10(8.79-10.63)/g). A genetic case (GSS-P102L) yielded a similar level of seeding activity in an autopsy brain sample. The range of PrPSc concentrations in the samples, determined by dot-blot assay, was 0.6-5.4 μg/g brain; therefore, we estimated that 1 SD50 unit was equivalent to 0.06-0.27 fg of PrPSc. The SD50 values of the affected brains dropped more than three orders of magnitude after autoclaving at 121°C. This new method for quantitation of human prion activity provides a new way to reduce the risk of iatrogenic prion transmission.
authors.affiliation: Department of Molecular Microbiology and Immunology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan.
authors.forename: Hanae
authors.initials: H
authors.lastname: Takatsuki
authors.affiliation: Department of Molecular Microbiology and Immunology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan.
authors.forename: Katsuya
authors.initials: K
authors.lastname: Satoh
authors.affiliation: Department of Physiology and Pharmacology, Faculty of Pharmaceutical Sciences, Fukuoka University, Fukuoka, Japan.
authors.forename: Kazunori
authors.initials: K
authors.lastname: Sano
authors.affiliation: Department of Molecular Microbiology and Immunology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Fuse
authors.affiliation: Department of Molecular Microbiology and Immunology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan.
authors.forename: Takehiro
authors.initials: T
authors.lastname: Nakagaki
authors.affiliation: Department of Molecular Microbiology and Immunology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan.
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Mori
authors.affiliation: Department of Molecular Microbiology and Immunology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Ishibashi
authors.affiliation: Department of neurology, Institute of Brain and Blood Vessels, Mihara Memorial Hospital, Isesaki, Japan.
authors.forename: Ban
authors.initials: B
authors.lastname: Mihara
authors.affiliation: Department of neurology, Institute of Brain and Blood Vessels, Mihara Memorial Hospital, Isesaki, Japan; Department of Neurology International Medical Center, Saitama Medical University, Saitama, Japan.
authors.forename: Masaki
authors.initials: M
authors.lastname: Takao
authors.affiliation: Department of Neuropathology, Institute for Medical Science of Aging, Aichi Medical University, Aichi, Japan.
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Iwasaki
authors.affiliation: Department of Neuropathology, Institute for Medical Science of Aging, Aichi Medical University, Aichi, Japan.
authors.forename: Mari
authors.initials: M
authors.lastname: Yoshida
authors.affiliation: Department of Molecular Microbiology and Immunology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan.
authors.forename: Ryuichiro
authors.initials: R
authors.lastname: Atarashi
authors.affiliation: Department of Molecular Microbiology and Immunology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan.
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Nishida
mesh_terms: D000328:Adult; D000368:Aged; D000682:Amyloid / Q000378:metabolism*; D001921:Brain / Q000378:metabolism* / Q000473:pathology; D007562:Creutzfeldt-Jakob Syndrome / Q000175:diagnosis / Q000378:metabolism; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D018620:PrPSc Proteins / Q000378:metabolism; D017096:Prion Diseases / Q000175:diagnosis / Q000235:genetics / Q000378:metabolism*; D011328:Prions / Q000378:metabolism
chemical_list: D000682:Amyloid; D018620:PrPSc Proteins; D011328:Prions