The real-time quaking-induced conversion assay for detection of human prion disease and study of other protein misfolding diseases

Developers

Matthias Schmitz, Maria Cramm, Byron Caughey, Inga Zerr, etc.

Description of the technology

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 in human brain and cerebrospinal fluid samples.

This technology is a fast and efficient protein amplification technique, real-time quaking-induced conversion, for the detection of abnormally folded prion protein scrapie seed in human brain and cerebrospinal fluid. In contrast to other in vitro misfolded protein amplification assays-such as protein misfolding cyclic amplification-which are based on sonication, the real-time quaking-induced conversion 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 real-time quaking-induced conversion 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.

Practical application

The technology, based on real-time quaking-induced conversion assay reactions in 96-well plates in an automated assay, permits the high-throughput analyses of samples and increases the potential for numerous other applications in scientific and industrial fields. The real-time quaking-induced conversion assay have been developed for most prion strains and many human and animal tissues and body fluids, which makes this method applicable for diagnostics, biological science, prescreening for potential therapeutics and testing of materials for contamination with infectious prions. The examples of the manifold applications of this method include human prion disease diagnostics in cerebrospinal fluid and in nasal brushings; prion strain typing, etc.

Laboratories

• Department of Neurology, University Medical Center Göttingen and German Center for Neurodegenerative Diseases (DZNE), Göttingen (Germany)
• Department of Medicine, The University of Melbourne, Parkville (Australia)
• Department of Molecular Microbiology and Immunology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki (Japan)
• Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Hamilton (USA)

Links

Publications

• Schmitz, M. et al. «The real-time quaking-induced conversion assay for detection of human prion disease and study of other protein misfolding diseases." 11 Nature Protocols (2016): 2233–2242.
• Schmitz, M. et al. «Application of an in vitro-amplification assay as a novel pre-screening test for compounds inhibiting the aggregation of prion protein scrapie." 6 Sci. Rep. (2016): 28711.
• Schmitz, M. et al. «Validation of 14-3-3 protein as a marker in sporadic Creutzfeldt-Jakob diagnostic." 53 Mol. Neurobiol. (2016): 2189–2199.