Rapid end-point quantitation of prion seeding activity with sensitivity comparable to bioassays

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 prpc 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 (sd50). brain tissue from 263k scrapie-affected hamsters gave sd50 values of 1011-1012/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 sd50 values of 103.5-105.7/ml,showing that nasal cavities release substantial prion infectivity that can be rapidly detected. cerebral spinal fluid from 263k scrapieaffected hamsters contained prion sd50 values of 102.0-102.9/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.