Prions are infectious agents that cause the inevitably fatal transmissible spongiform encephalopathy (TSE) in animals and humans(9,18). The prion protein has two distinct isoforms, the non-infectious host-encoded protein (PrP(C)) and the infectious protein (PrP(Sc)), an abnormally-folded isoform of PrP(C 8). One of the challenges of working with prion agents is the long incubation period prior to the development of clinical signs following host inoculation(13). This traditionally mandated long and expensive animal bioassay studies. Furthermore, the biochemical and biophysical properties of PrP(Sc) are poorly characterized due to their unusual conformation and aggregation states. PrP(Sc) can seed the conversion of PrP(C) to PrP(Sc) in vitro(14). PMCA is an in vitro technique that takes advantage of this ability using sonication and incubation cycles to produce large amounts of PrP(Sc), at an accelerated rate, from a system containing excess amounts of PrP(C) and minute amounts of the PrP(Sc) seed(19). This technique has proven to effectively recapitulate the species and strain specificity of PrP(Sc) conversion from PrP(C), to emulate prion strain interference, and to amplify very low levels of PrP(Sc) from infected tissues, fluids, and environmental samples(6,7,16,23) . This paper details the PMCA protocol, including recommendations for minimizing contamination, generating consistent results, and quantifying those results. We also discuss several PMCA applications, including generation and characterization of infectious prion strains, prion strain interference, and the detection of prions in the environment.
All Science Journal Classification (ASJC) codes
- Chemical Engineering(all)
- Biochemistry, Genetics and Molecular Biology(all)
- Immunology and Microbiology(all)