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

<div><p>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 (PrP^d) in brain tissue biopsies and cerebral spinal fluid, has great potential to become a method for diagnosing prion disease <i>ante mortem</i>. 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 <i>in vitro</i>-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.</p></div

The effect of substrate concentration on RT-QuIC.

<p>RT-QuIC was performed using hamster rPrP substrate at concentrations of 20, 35, 45, and 60 µg/well. Reactions contained minimal salt (5.5 mM NaCl) and employed sCJD M/V brain homogenate (BH) diluted 10⁻⁴ to seed conversion.</p

The effect of batch variability and seed genotype on RT-QuIC.

<p>RT-QuIC was performed using two different batches, Batch #1 (A) and Batch #2 (B), of hamster rPrP. Both assays employed hamster rPrP at a concentration of 60 µg/well as the substrate and sCJD M/V brain homogenate (BH) at the indicated dilutions as the seed. (C) RT-QuIC was performed using hamster rPrP Batch #1 at a concentration of 60 µg/well as the substrate and sCJD M/M brain homogenate (BH) at indicated dilutions to seed conversion. Reactions contained minimal salt (5.5 mM NaCl).</p