Defining and Assessing Analytical Performance Criteria for Transmissible Spongiform Encephalopathy-Detecting Amyloid Seeding Assays

Transmissible spongiform encephalopathies (TSEs) are infectious, fatal neurodegenerative diseases that affect production animal health, and thus human food safety. Enhanced TSE detection methods mimic the conjectured basis for prion replication, in vitro; biological matrices can be tested for prion activity via their ability to convert recombinant cellular prion protein (PrP) into amyloid fibrils; fluorescent spectra changes of amyloid-binding fluorophores in the reaction vessel detect fibril formation. In vitro PrP conversion techniques have high analytical sensitivity for prions, comparable with that of bioassays, yet no such protocol has gained regulatory approval for use in animal TSE surveillance programs. This study describes a timed in vitro PrP conversion protocol with accurate, well-defined analytical criteria based on probability density and mass functions of TSE and TSE associated thioflavin T signal times, a new approach within this field. The prion detection model used is elk chronic wasting disease (CWD) in brain tissues. The protocol and analytical criteria proved as sensitive for elk CWD as two bioassay models, and upward of approximately 1.2 log10 more sensitive than the most sensitive TSE rapid test we assessed. Furthermore, we substantiate that timing in vitro PrP conversion may be used to titrate TSE infectivity, and, as a result, provide a comprehensive extrapolation of analytical sensitivity differences between bioassay, TSE rapid tests, and in vitro PrP conversion for elk CWD.