Sensitive detection of pathological prion protein by cyclic amplification of protein misfolding.
Prions are the infectious agents responsible for transmissible spongiform encephalopathies. The principal component of prions is the glycoprotein PrP(Sc), which is a conformationally modified isoform of a normal cell-surface protein called PrP(C) (ref. 1). During the time between infection and the appearance of the clinical symptoms, minute amounts of PrP(Sc) replicate by conversion of host PrP(C), generating large amounts of PrP(Sc) aggregates in the brains of diseased individuals. We aimed to reproduce this event in vitro. Here we report a procedure involving cyclic amplification of protein misfolding that allows a rapid conversion of large excess PrP(C) into a protease-resistant, PrP(Sc)-like form in the presence of minute quantities of PrP(Sc) template. In this procedure, conceptually analogous to polymerase chain reaction cycling, aggregates formed when PrP(Sc) is incubated with PrP(C) are disrupted by sonication to generate multiple smaller units for the continued formation of new PrP(Sc). After cyclic amplification more than 97% of the protease-resistant PrP present in the sample corresponds to newly converted protein. The method could be applied to diagnose the presence of currently undetectable prion infectious agent in tissues and biological fluids, and may provide a unique opportunity to determine whether PrP(Sc) replication results in the generation of infectivity in vitro.
Saborio
G P
GP
Serono Pharmaceutical Research Institute, Geneva, Switzerland. gabriela.saborio@serono.com
Permanne
B
B
Soto
C
C
D000818:Animals; D001923:Brain Chemistry; D002623:Chemistry Techniques, Analytical / Q000379:methods; D006224:Cricetinae; D018621:PrPC Proteins; D018620:PrPSc Proteins / Q000032:analysis* / Q000737:chemistry; D017096:Prion Diseases / Q000175:diagnosis / Q000209:etiology; D017510:Protein Folding; D051381:Rats; D012608:Scrapie / Q000378:metabolism; D013698:Templates, Genetic
D018621:PrPC Proteins; D018620:PrPSc Proteins