Abstract
Prions are the infectious agents responsible for transmissible spongiform encephalopathies. The principal component of prions is the glycoprotein PrPSc, which is a conformationally modified isoform of a normal cell-surface protein called PrPC (ref. 1). During the time between infection and the appearance of the clinical symptoms, minute amounts of PrPSc replicate by conversion of host PrPC, generating large amounts of PrPSc 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 PrPC into a protease-resistant, PrPSc-like form in the presence of minute quantities of PrPSc template. In this procedure, conceptually analogous to polymerase chain reaction cycling, 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-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 PrPSc replication results in the generation of infectivity in vitro.
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Acknowledgements
We thank S. Peano, A. Conz, L. Anderes and M.-J. Frossard for technical assistance and R. J. Kascsak for providing 3F4 anti-PrP antibody. We are grateful to M. Pocchiari, S. Fumero, T. Wells, K. Maundrell and J. DeLamarter for reading the manuscript and providing comments. We also thank C. Herbert for help in the preparation of the figures.
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Saborio, G., Permanne, B. & Soto, C. Sensitive detection of pathological prion protein by cyclic amplification of protein misfolding. Nature 411, 810â813 (2001). https://doi.org/10.1038/35081095
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DOI: https://doi.org/10.1038/35081095
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