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Background Bacterially-produced recombinant prion protein (rPrP) has traditionally been used for in vitro fibrillation assays and reagent development for prion disease research. In recent years, it has also been used as a substrate for real-time quaking-induced conversion (RT-QuIC), a very sensitive method of detecting the presence of the misfolded, disease-associated isoform of the prion protein (PrP d ). Multi-centre trials have demonstrated that RT-QuIC is a suitably reliable and robust technique for clinical practice; however, in the absence of a commercial supplier of rPrP as a substrate for RT-QuIC, laboratories have been required to independently generate this key component of the assay. No harmonized method for producing the protein has been agreed upon, in part due to the variety of substrates that have been applied in RT-QuIC. Methods This study examines the effects of two different rPrP refolding protocols on the production, QuIC performance, and structure characteristics of two constructs of rPrP commonly used in QuIC: full length hamster and a sheep-hamster chimeric rPrP. Results Under the described conditions, the best performing substrate was the chimeric sheep-hamster rPrP produced by shorter guanidine-HCl exposure and faster gradient elution. Conclusions The observation that different rPrP production protocols influence QuIC performance indicates that caution should be exercised when comparing inter-laboratory QuIC results.

The Prion Laboratory Section of the Public Health Agency of Canada supports heath care professionals dealing with patients suspected to have Creutzfeldt-Jakob disease (CJD) by testing cerebrospinal fluid (CSF) for protein markers of CJD. To better serve Canadian diagnostic requirements, a quaking-induced conversion (QuIC)-based assay has been added to the test panel. The QuIC tests exploit the ability of disease-associated prion protein, found in the CSF of a majority of CJD patients, to convert a recombinant prion protein (rPrP) into detectable amounts of a misfolded, aggregated form of rPrP. The rPrP aggregates interact with a specific dye, causing a measurable change in the dye's fluorescence emission spectrum. Optimal test and analysis parameters were empirically determined. Taking both practical and performance considerations into account, an endpoint QuIC (EP-QuIC) configuration was chosen. EP-QuIC uses a thermo-mixer to perform the shaking necessary to produce the quaking-induced conversions. Fluorescence readings are obtained from a microwell fluorescence reader only at the beginning and the end of EP-QuIC reactions. Samples for which the relative fluorescence unit ratio between the initial and final readings represent a ≥4 increase in signal intensity in at least two of the three replicates are classified as positive. A retrospective analysis of 91 CSF samples that included 45 confirmed cases of CJD and 46 non-CJD cases was used to estimate the performance characteristics of the EP-QuIC assay. The diagnostic sensitivity and specificity of the EP-QuIC test of this set of samples were 98 and 91%, respectively.

Transmissible spongiform encephalopathies (TSEs) are neurodegenerative disorders caused by the presence of an infectious prion protein. The primary site of pathology is the brain characterized by neuroinflammation, astrogliosis, prion fibrils, and vacuolation. The events preceding the observed pathology remain in question. We sought to identify biomarkers in the brain of TSE-infected and aged-matched control mice using two-dimensional fluorescence difference gel electrophoresis (2D-DIGE). Since the brain proteome is too complex to resolve all proteins using 2D-DIGE, protein samples are initially filtered through either concanavalin A (ConA) or wheat-germ agglutinin (WGA) columns. Four differentially abundant proteins are identified through screening of the two different glycoproteomes: Neuronal growth regulator 1 (NEGR1), calponin-3 (CNN3), peroxiredoxin-6 (Prdx6), and glial fibrillary acidic protein (GFAP). Confirmatory Western blots are performed with samples from TSE-infected and comparative Alzheimer's disease (AD) affected brains and their respective controls from time points throughout the disease courses. The abundance of three of the four proteins increases significantly during later stages of prion disease whereas NEGR1 decreases in abundance. Comparatively, no significant changes are observed in later stages of AD. Our lab is the first to associate the glycosylated NEGR1 protein with prion disease pathology.