Highly Efficient Amplification of Chronic Wasting Disease Agent by Protein Misfolding Cyclic Amplification with Beads (PMCAb)

About the Authors:

Chad J. Johnson

Affiliation: Environmental Chemistry and Technology Program and Department of Soil Science, University of Wisconsin, Madison, Wisconsin, United States of America

Judd M. Aiken

Affiliation: Center for Prions and Protein Misfolding Diseases, University of Alberta, Edmonton, Alberta, Canada

Debbie McKenzie

Affiliation: Center for Prions and Protein Misfolding Diseases, University of Alberta, Edmonton, Alberta, Canada

Michael D. Samuel

Affiliation: U.S. Geological Survey, Wisconsin Cooperative Wildlife Research Unit, University of Wisconsin, Madison, Wisconsin, United States of America

Joel A. Pedersen

* E-mail: [email protected]

Affiliation: Environmental Chemistry and Technology Program and Department of Soil Science, University of Wisconsin, Madison, Wisconsin, United States of America

Introduction

Transmissible spongiform encephalopathies (TSEs) or prion diseases are a class of rare progressive neurodegenerative diseases and include scrapie in sheep, bovine spongiform encephalopathy in cattle, and chronic wasting disease (CWD) in deer, elk and moose, and Creutzfeldt-Jakob disease in humans. The etiological agents in TSEs lack specific nucleic acid and are referred to as prions. Substantial experimental evidence indicates that prions are composed primarily, if not solely, of a disease-associated conformer of the host-encoded prion protein (PrP) [1], [2]. The central biochemical event in the propagation of prion diseases is the refolding of the normal cellular prion protein (PrP^C) into a disease-associated conformer (PrP^TSE), a substantial fraction of which partially resists digestion by proteinase K (PK) and is denoted PrP^res. The precise molecular mechanism of PrP^C-to-PrP^TSE conversion is unknown. Template-assisted refolding and nucleation-polymerization models have been advanced as mechanisms [3], [4]. The multiple stable conformations of PrP^TSE lead to the phenomenon of a single protein being able to encipher multiple strains.

Protein misfolding cyclic amplification (PMCA) [5] has emerged as an important technique for detecting low levels of PrP^res in biological samples [6]–[8]. PMCA exploits the ability of PrP^TSE to catalyze the conversion of PrP^C to the misfolded isoform in a manner conceptually similar to the polymerase chain reaction. The amount of PrP^res is increased by successive cycles of sonication (to disrupt PrP^TSE aggregates) and amplification using PrP^C from brain homogenate as a substrate. Sensitivity can be increased by replenishing the substrate after a round of PMCA (one round consists of...