Quaking-Induced Conversion Assays for the Detection and Diagnosis of Prion Diseases

Abstract

The seeding activity of prions has been exploited for the development of a number of ultrasensitive assays for transmissible spongiform encephalopathies (TSEs). Among the more practical assays are those that use recombinant PrP^Sen (rPrP^Sen) as a substrate for prion-seeded conversion into amyloid fibrils, shaking rather than sonication, and fluorescence detection in multiwell plates. These include the amyloid seeding assay (ASA), real-time QuIC (RT-QuIC), and enhanced QuIC (eQuIC). Recent applications of RT-QuIC to the antemortem diagnosis of sporadic Creutzfeldt–Jakob disease (sCJD) using cerebrospinal fluid (CSF) showed improved specificity (nearly 100%) relative to assays for other CSF markers. Moreover, the RT-QuIC can be quantitative and as sensitive as animal bioassays, allowing measurements of prion seeding activity in CSF and nasal fluids. In hamster CSF, the time course of seeding activity accumulation can vary markedly with route of scrapie inoculation. To enhance sensitivity and to cope with inhibitor-laden sample types such as blood plasma, an immune capture step was integrated with RT-QuIC, giving rise to the eQuIC assay. eQuIC can detect up to 10¹⁴-fold dilutions of human CJD brain homogenate in blood plasma and discriminate plasma samples from scrapie-infected and uninfected rodents. Although further work is required to fully validate various applications of RT-QuIC, eQuIC, and related tests, these assays improve prospects for practical prion detection in humans, animals, biomaterials, and the environment.