Abstract
The development and adaption of in vitro misfolded protein amplification systems has been a major innovation in the detection of abnormally folded prion protein scrapie (PrPSc) in human brain and cerebrospinal fluid (CSF) samples. Herein, we describe a fast and efficient protein amplification technique, real-time quaking-induced conversion (RT-QuIC), for the detection of a PrPSc seed in human brain and CSF. In contrast to other in vitro misfolded protein amplification assays—such as protein misfolding cyclic amplification (PMCA)—which are based on sonication, the RT-QuIC technique is based on prion seed–induced misfolding and aggregation of recombinant prion protein substrate, accelerated by alternating cycles of shaking and rest in fluorescence plate readers. A single RT-QuIC assay typically analyzes up to 32 samples in triplicate, using a 96-well-plate format. From sample preparation to analysis of results, the protocol takes ∼87 h to complete. In addition to diagnostics, this technique has substantial generic analytical applications, including drug screening, prion strain discrimination, biohazard screening (e.g., to reduce transmission risk related to prion diseases) and the study of protein misfolding; in addition, it can potentially be used for the investigation of other protein misfolding diseases such as Alzheimer's and Parkinson's disease.
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Acknowledgements
This study was performed as part of the Clinical Dementia Center at the University Medical Center Göttingen and was partly supported by grants from the EU Joint Program—Neurodegenerative Disease Research (JPND-DEMTEST (Biomarker-based diagnosis of rapid progressive dementias—optimization of diagnostic protocols, 01ED1201A) and by the Robert Koch Institute through funds from the Federal Ministry of Health (grant no. 1369-341). This work was also supported in part by the Intramural Research Program of the NIAID. S.C. is supported by a NHMRC Practitioner Fellowship (identification no. APP1005816) and by the Australian National Creutzfeldt-Jakob Disease Registry (ANCJDR), which is funded by the Commonwealth Department of Health.
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M.S. was involved in study concept and design, and validated the protocol, interpreted data and wrote the manuscript. M.C. performed experiments, analyzed and interpreted data, prepared figures and wrote the manuscript. F.L. provided samples and interpreted data.
D.M.-C. designed Figure 1. S.C., R.A., K.S., C.D.O., B.R.G., S.Z. and B.C. critically revised the manuscript. W.J.S.-S. provided samples. I.Z. supervised the study and critically revised the manuscript.
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Schmitz, M., Cramm, M., Llorens, F. et al. The real-time quaking-induced conversion assay for detection of human prion disease and study of other protein misfolding diseases. Nat Protoc 11, 2233–2242 (2016). https://doi.org/10.1038/nprot.2016.120
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DOI: https://doi.org/10.1038/nprot.2016.120
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