Sensitive detection of pathological prion protein by cyclic amplification of protein misfolding

Prions are the infectious agents responsible for transmissible spongiform e ncephalopathies. The principal component of prions is the glycoprotein PrPS c, which is a conformationally modified isoform of a normal cell-surface pr otein called PrPC (ref. 1). During the time between infection and the appea rance of the clinical symptoms, minute amounts of PrPSc replicate by conver sion of host PrPC, generating large amounts of PrPSc aggregates in the brai ns 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 PrPC into a protease-resist ant, PrPSc-like form in the presence of minute quantities of PrPSc template . In this procedure, conceptually analogous to polymerase chain reaction cy cling, aggregates formed when PrPSc is incubated with PrPC are disrupted by sonication to generate multiple smaller units for the continued formation of new PrPSc. After cyclic amplification more than 97% of the protease-resi stant 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 PrPSc replication results in the g eneration of infectivity in vitro.