title: Rapid end-point quantitation of prion seeding activity with sensitivity comparable to bioassays.
abstract: A major problem for the effective diagnosis and management of prion diseases is the lack of rapid high-throughput assays to measure low levels of prions. Such measurements have typically required prolonged bioassays in animals. Highly sensitive, but generally non-quantitative, prion detection methods have been developed based on prions' ability to seed the conversion of normally soluble protease-sensitive forms of prion protein to protease-resistant and/or amyloid fibrillar forms. Here we describe an approach for estimating the relative amount of prions using a new prion seeding assay called real-time quaking induced conversion assay (RT-QuIC). The underlying reaction blends aspects of the previously described quaking-induced conversion (QuIC) and amyloid seeding assay (ASA) methods and involves prion-seeded conversion of the alpha helix-rich form of bacterially expressed recombinant PrP(C) to a beta sheet-rich amyloid fibrillar form. The RT-QuIC is as sensitive as the animal bioassay, but can be accomplished in 2 days or less. Analogous to end-point dilution animal bioassays, this approach involves testing of serial dilutions of samples and statistically estimating the seeding dose (SD) giving positive responses in 50% of replicate reactions (SD(50)). Brain tissue from 263K scrapie-affected hamsters gave SD(50) values of 10(11)-10(12)/g, making the RT-QuIC similar in sensitivity to end-point dilution bioassays. Analysis of bioassay-positive nasal lavages from hamsters affected with transmissible mink encephalopathy gave SD(50) values of 10(3.5)-10(5.7)/ml, showing that nasal cavities release substantial prion infectivity that can be rapidly detected. Cerebral spinal fluid from 263K scrapie-affected hamsters contained prion SD(50) values of 10(2.0)-10(2.9)/ml. RT-QuIC assay also discriminated deer chronic wasting disease and sheep scrapie brain samples from normal control samples. In principle, end-point dilution quantitation can be applied to many types of prion and amyloid seeding assays. End point dilution RT-QuIC provides a sensitive, rapid, quantitative, and high throughput assay of prion seeding activity.
authors.affiliation: Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Disease, Hamilton, Montana, United States of America.
authors.forename: Jason M
authors.initials: JM
authors.lastname: Wilham
authors.forename: Christina D
authors.initials: CD
authors.lastname: Orrú
authors.forename: Richard A
authors.initials: RA
authors.lastname: Bessen
authors.forename: Ryuichiro
authors.initials: R
authors.lastname: Atarashi
authors.forename: Kazunori
authors.initials: K
authors.lastname: Sano
authors.forename: Brent
authors.initials: B
authors.lastname: Race
authors.forename: Kimberly D
authors.initials: KD
authors.lastname: Meade-White
authors.forename: Lara M
authors.initials: LM
authors.lastname: Taubner
authors.forename: Andrew
authors.initials: A
authors.lastname: Timmes
authors.forename: Byron
authors.initials: B
authors.lastname: Caughey
mesh_terms: D000682:Amyloid / Q000032:analysis; D000818:Animals; D001921:Brain; D002555:Cerebrospinal Fluid / Q000737:chemistry; D006224:Cricetinae; D003670:Deer; D023381:Endpoint Determination / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods / Q000592:standards*; D006801:Humans; D008722:Methods; D008907:Mink; D011328:Prions / Q000032:analysis*; D017433:Protein Structure, Secondary; D012756:Sheep
chemical_list: D000682:Amyloid; D011328:Prions