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Ultrasensitive detection of scrapie prion protein using seeded conversion of recombinant prion protein

Abstract

The scrapie prion protein isoform, PrPSc, is a prion-associated marker that seeds the conformational conversion and polymerization of normal protease-sensitive prion protein (PrP-sen). This seeding activity allows ultrasensitive detection of PrPSc using cyclical sonicated amplification (PMCA) reactions and brain homogenate as a source of PrP-sen. Here we describe a much faster seeded polymerization method (rPrP-PMCA) which detects ≥50 ag of hamster PrPSc (≈0.003 lethal dose) within 2–3 d. This technique uses recombinant hamster PrP-sen, which, unlike brain-derived PrP-sen, can be easily concentrated, mutated and synthetically tagged. We generated protease-resistant recombinant PrP fibrils that differed from spontaneously initiated fibrils in their proteolytic susceptibility and by their infrared spectra. This assay could discriminate between scrapie-infected and uninfected hamsters using 2-μl aliquots of cerebral spinal fluid. This method should facilitate the development of rapid, ultrasensitive prion assays and diagnostic tests, in addition to aiding fundamental studies of structure and mechanism of PrPSc formation.

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Figure 1: Comparison of HaPrPSc-seeded and unseeded rHaPrP-sen conversion reactions.
Figure 2: Detection limit of rPrP-PMCA.
Figure 3: Seeding competition between rHaPrP-res(Sc) and rHaPrP-res(spon).
Figure 4: Seeding rPrP-PMCA with CSF.

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Acknowledgements

We thank K. Meade-White and B. Race for providing us with the CSF samples, G. Raymond for technical assistance, and G. Baron, B. Race and Y. Taguchi for helpful discussions and critical assessment of the manuscript. This research was funded by the Intramural Research Program of the US National Institute of Allergy and Infectious Diseases. V.L.S. also receives funding from a Clinical Fellowship Award from the Alberta Heritage Foundation for Medical Research.

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Contributions

R.A. initiated the project, conceived and performed most experiments, and helped write the manuscript; R.A.M. prepared rPrP-sen, performed FTIR and edited the manuscript; V.L.S. performed electron microscopy and edited the manuscript; A.G.H. performed supporting experiments and wrote the step-by-step protocol; D.W.D. performed electron microscopy; H.A.O. performed supporting experiments; S.A.P. supervised rPrP-sen preparation and edited the manuscript; B.C. coordinated the project and wrote the manuscript.

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Correspondence to Byron Caughey.

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The authors declare no competing financial interests.

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Supplementary Figures 1–5, Supplementary Methods (PDF 701 kb)

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Atarashi, R., Moore, R., Sim, V. et al. Ultrasensitive detection of scrapie prion protein using seeded conversion of recombinant prion protein. Nat Methods 4, 645–650 (2007). https://doi.org/10.1038/nmeth1066

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