Rapid and Quantitative Assay of Amyloid-Seeding Activity in Human Brains Affected with Prion Diseases

The infectious agents of the transmissible spongiform encephalopathies are composed of amyloidogenic prion protein, PrP(Sc). Real-time quaking-induced conversion can amplify very small amounts of PrP(Sc) 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 (SD(50)) 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 PrP(Sc) concentrations in the samples, determined by dot-blot assay, was 0.6–5.4 μg/g brain; therefore, we estimated that 1 SD(50) unit was equivalent to 0.06–0.27 fg of PrP(Sc). The SD(50) 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.