Protein misfolding cyclic amplification of infectious prions

• Published in: Nature protocols Vol. 7; no. 7; pp. 1397 - 1409
• Main Authors: Morales, Rodrigo, Duran-Aniotz, Claudia, Diaz-Espinoza, Rodrigo, Camacho, Manuel V, Soto, Claudio
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 28.06.2012; Nature Publishing Group
• Subjects: 631/1647/2196; 64; 64/110; 692/699/375/1937; 82; Alzheimer's disease; Amplification; Analytical Chemistry; Biological Techniques; Blood; Blood & organ donations; Brain research; Computational Biology/Bioinformatics; Disease; Disease transmission; Food industry; Food safety; Humans; In vivo methods and tests; Infectivity; Life Sciences; Microarrays; Organic Chemistry; Polymers; Prion Diseases - genetics; Prion protein; Prions; Prions - chemistry; Protein Engineering - methods; Protein Folding; Protein Multimerization; Proteins; Protocol; Replication; Seeds; Sonication; Sonication - methods; Ultrasonic imaging; Veterinary diseases
• ISSN: 1754-2189; 1750-2799; 1750-2799
• DOI: 10.1038/nprot.2012.067

Prions are proteinaceous infectious agents responsible for the transmission of prion diseases. The lack of a procedure for cultivating prions in the laboratory has been a major limitation to the study of the unorthodox nature of this infectious agent and the molecular mechanism by which the normal prion protein (PrP C ) is converted into the abnormal isoform (PrP Sc ). Protein misfolding cyclic amplification (PMCA), described in detail in this protocol, is a simple, fast and efficient methodology to mimic prion replication in the test tube. PMCA involves incubating materials containing minute amounts of infectious prions with an excess of PrP C and boosting the conversion by cycles of sonication to fragment the converting units, thereby leading to accelerated prion replication. PMCA is able to detect the equivalent of a single molecule of infectious PrP Sc and propagate prions that maintain high infectivity, strain properties and species specificity. A single PMCA assay takes little more than 3 d to replicate a large amount of prions, which could take years in an in vivo situation. Since its invention 10 years ago, PMCA has helped to answer fundamental questions about this intriguing infectious agent and has been broadly applied in research areas that include the food industry, blood bank safety and human and veterinary disease diagnosis.