Abstract

The infectious agents of the transmissible spongiform encephalopathies are composed of amyloidogenic prion protein, PrPSc. Real-time quaking-induced conversion can amplify very small amounts of PrPSc seeds in tissues/body fluids of patients or animals. Using this in vitro PrP-amyloid amplification assay, we quantitated the seeding activity of affected human brains. End-point assay using serially diluted brain homogenates of sporadic Creutzfeldt–Jakob disease patients demonstrated that 50% seeding dose (SD50) is reached approximately 1010/g brain (values varies 108.79–10.63/g). A genetic case (GSS-P102L) yielded a similar level of seeding activity in an autopsy brain sample. The range of PrPSc concentrations in the samples, determined by dot-blot assay, was 0.6–5.4 μg/g brain; therefore, we estimated that 1 SD50 unit was equivalent to 0.06–0.27 fg of PrPSc. The SD50 values of the affected brains dropped more than three orders of magnitude after autoclaving at 121°C. This new method for quantitation of human prion activity provides a new way to reduce the risk of iatrogenic prion transmission.

Highlights

  • Human prion diseases (HPD) are neurodegenerative diseases caused by accumulation of amyloidogenic prion protein (PrPSc), which is generated from the cellular prion protein (PrPC) via a conformational change

  • The fluorescence of ThT was elevated at dilutions from 5×10–5 to 5×10–8, at 5×10–9 dilution there was yielded no reaction, as did a negative controls (S1 Fig)

  • We were successfully able to analyze eight other sporadic Creutzfeldt— Jakob disease (CJD) and a genetic case, Gerstmann—Sträussler—Scheinker syndrome (GSS)-P102L; in all of these cases, the SD50 values were similar (Figs 1b, 2 and Table 1)

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Summary

Introduction

Human prion diseases (HPD) are neurodegenerative diseases caused by accumulation of amyloidogenic prion protein (PrPSc), which is generated from the cellular prion protein (PrPC) via a conformational change. PrPSc is detected in neuronal tissues, and in lymphoid tissues (e.g., spleen, tonsil, lymph node) [1, 2] and muscles of some sporadic Creutzfeldt— Jakob disease (CJD) (sCJD) patients [1]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

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