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 (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 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 SD₅₀ 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. Prion diseases are deadly infectious neurodegenerative disorders of mammals which involve the misfolding of host prion protein. To better manage these diseases, we need to be able to detect and quantify the infectious particles, or prions, in biological samples. However, current tests lack the sensitivity, speed and/or quantitative capabilities required for many important applications in medicine, agriculture, wildlife biology and research. To address this problem, we have developed a new prion assay that is highly sensitive, rapid, and quantitative. This assay takes advantage of the ability of miniscule amounts of infectious prions to seed the misfolding of large excesses of normal prion protein in test tube reactions. Quantitation is achieved by testing a range of sample dilutions and determining loss of seeding activity, i.e. the end-point dilution. Similar analyses have long been used to quantify prions by inoculation into animals; however, such bioassays take months or years to perform and are both animal-intensive and expensive. Our new method provides a more practical means of detecting and quantifying prions. So far, we have applied this assay to prions from sheep, deer, and hamsters, and have found surprisingly high levels of prions in the nasal and cerebral spinal fluids of infected hamsters.