Exploring Physical and Chemical Factors Influencing the Properties of Recombinant Prion Protein and the Real-Time Quaking-Induced Conversion (RT-QuIC) Assay

et al. (2014) Exploring Physical and Chemical Factors Influencing the Properties of Recombinant Prion Protein and the Real-Time Quaking-Induced Conversion (RT-QuIC) Assay. PLoS ONE 9(1): e84812. doi:10.1371/journal.pone.0084812 Exploring Physical and Chemical Factors Influencing the Properties of Recombinant Prion Protein and the Real- Time Quaking-Induced Conversion (RT-QuIC) Assay David Knox 0 Keding Cheng 0 Angela Sloan 0 Kristen M. Avery 0 Michael Coulthart 0 Michael Carpenter 0 J. 0 Andrew Francis Hill, University of Melbourne, Australia 0 1 National Microbiology Laboratory, Public Health Agency of Canada , Winnipeg, Manitoba , Canada , 2 Department of Human Anatomy and Cell Sciences, Faculty of Medicine, University of Manitoba , Winnipeg, Manitoba , Canada , 3 Centre for Food-borne, Environmental and Zoonotic Infectious Diseases, Public Health Agency of Canada , Ottawa, Ontario , Canada , 4 Department of Medical Microbiology, Faculty of Medicine, University of Manitoba , Winnipeg, Manitoba , Canada Real-time quaking-induced conversion (RT-QuIC), a highly specific and sensitive assay able to detect low levels of the disease-inducing isoform of the prion protein (PrPd) in brain tissue biopsies and cerebral spinal fluid, has great potential to become a method for diagnosing prion disease ante mortem. In order to standardize the assay method for routine analysis, an understanding of how physical and chemical factors affect the stability of the recombinant prion protein (rPrP) substrate and the RT-QuIC assay's sensitivity, specificity, and reproducibility is required. In this study, using sporadic Creutzfeldt-Jakob Disease brain homogenate to seed the reactions and an in vitro-expressed recombinant prion protein, hamster rPrP, as the substrate, the following factors affecting the RT-QuIC assay were examined: salt and substrate concentrations, substrate storage, and pH. Results demonstrated that both the generation of the quality and quantities of rPrP substrate critical to the reaction, as well as the RT-QuIC reaction itself required strict adherence to specific physical and chemical conditions. Once optimized, the RT-QuIC assay was confirmed to be a very specific and sensitive assay method for sCJD detection. Findings in this study indicate that further optimization and standardization of RT-QuIC assay is required before it can be adopted as a routine diagnostic test. - Prion diseases, such as Creutzfeldt-Jakob Disease (CJD), are invariably fatal degenerative syndromes of the central nervous system. The central event of the disease process is the seeded conversion of host-encoded cellular prion protein (PrPc) into a misfolded disease-associated isoform (PrPd). Currently, definitive diagnoses rely upon the detection of PrPd in a brain biopsy or post mortem brain tissue. The extremely low amounts of PrPd present in other tissues make ante mortem tests based on its presence problematic. One solution to this challenge has been the development of techniques that exploit the ability of PrPd to seed the conformational conversion of a PrPc substrate in vitro. Real-time quakinginduced conversion (RT-QuIC) is an example of a technique that utilizes minute amounts of PrPd present in the test sample to seed the conformational conversion of normal soluble recombinant prion protein (rPrPc) from a highly a-helical structure into an amyloid fibril that is rich in b-sheet [1]. The technique has proven successful for sensitive prion detection in the 263K hamster scrapie model and in human CJD patient samples [25]. Utilizing brain homogenate (BH) or cerebral spinal fluid (CSF) samples, RTQuIC has shown higher specificity than does 14-3-3 testing for sCJD [3,6,7] and specificity comparable to post-mortem immunohistochemistry of brain tissue [3,6]. Hence, RT-QuIC has been shown to be a specific and sensitive method for PrPd detection, with the potential to become the state-of-the-art ante-mortem clinical test for the diagnosis of sporadic Creutzfeldt-Jakob disease (sCJD) [2,3]. Before clinical application of RT-QuIC can be widely adopted, the assay must be standardized for optimal analytical performance. Currently, users of the technique employ various buffer conditions and diverse fluorescence reading outputs [3,6]. In addition, acidic pH as well as higher salt and detergent concentrations have been shown to either enhance RT-QuIC reactions or result in spontaneous aggregation of the recombinant protein substrate [3]. To establish optimal RT-QuIC reaction conditions in our laboratory a commercially available CJD brain homogenate (National Institute for Biological Standards and Control, NIBSC), previously confirmed by Western blot to contain high levels of proteinase K resistant PrP, was utilised as the test seed. This seed, in combination with an in-house generated recombinant fulllength hamster PrP (rPrP) as the substrate, was used to explore the physical and chemical factors affecting RT-QuIC. The parameters of the optimal reaction conditions, such as salt, pH, and substrate concentrations, were determined, as well as the effects of elution method, dialysis conditions, and storage on substrate quality. Optimization of rPrP Purification Procedure In order to increase the yield of rPrP, slight modifications to published methods were made [4,5]. First, slower rPrP refolding was applied (approximately 18 hours on an HPLC system). Following refolding, the column was transferred to an FLPC system and isocratic elution (500 mM imidazole, 100 mM sodium phosphate and 10 mM Tris-HCl, pH 5.8), as opposed to gradient elution, was performed. The generated eluate appeared cloudy and filtration using a 0.22 mm syringe filter was difficult. Gelstaining demonstrated that the amount of rPrP collected in the syringe-filter filtrate was small compared to the large amount of rPrP in the eluate (data not shown). This suggested that the 0.22 mm filter was blocking the passage of rPrP aggregates. Using these conditions, the concentration of rPrP in the filtrate was too low to proceed with RT-QuIC trials. When 7.5 mL fractions of eluate were collected into tubes containing 2.5 mL of 10 mM sodium phosphate (pH 5.8), the eluate (final concentration: 375 mM imidazole, 77.5 mM sodium phosphate, and 7.5 mM Tris-HCl, pH 5.8) appeared clear and the mixture was easily filtered by a 0.22 mm filter before dialysis against 10 mM sodium phosphate (5.8). This step of immediately diluting the eluate was critical for the production of sufficient amounts of RT-QuIC compatible rPrP. Determination of Optimal RT-QuIC Conditions In order to replicate previously published conditions [4,5] rPrP was added to reaction mixtures that consisted of EDTA (1 mM), thioflavin T (ThT, 10 mM), and 1X phosphate buffered saline (10 mM sodium phosphate, 138 mM NaCl, and 2.7 mM KCl). In addition to the 138 mM NaCl contributed by PBS, many laboratories add additional NaCl to the reaction mixture, resulting in final NaCl concentratio (...truncated)