Protein Structure and Biology
نویسندگان
چکیده
The ability of PrP to convert PrP into protease-resistance isoforms has been exploited using a variety of techniques such as protein misfolding cyclic amplification (PMCA), quaking induced conversion (QuIC) and most recently, real-time quaking induced conversion (RT-QuIC). These cell-free assays have enabled a better understanding of prion diseases and have facilitated the development of potential diagnostic tests for prionrelated diseases. The RT-QuIC technique exploits the ability of PrP in brain tissue or CSF to induce a recombinant PrP to change shape and aggregate over time. This aggregation is observed by the binding of Thioflavin T (ThT) in the reaction mixture to the aggregates causing a change in the ThT emission spectrum, which can be monitored in real time. Studies have shown that CSF samples from hamsters inoculated with experimental scrapie and from patients with sCJD can be correctly identified using RT-QuIC. At the National CJD Research and Surveillance unit (NCJDRSU) we have completed a retrospective study and are currently undertaking a prospective audit investigating the value of RT-QuIC in the diagnosis of sCJD. During our studies various elements of the technique have been modified during the optimisation process of this potential sCJD CSF diagnostic tool such as: the volume of CSF; the source of recombinant PrP (rPrP); the size of construct (Full-length or Truncated); and the shaking time, speed and mechanism used, each can make a significant difference to the results obtained. This study looks at some of these variables and what the effect of altering them has on the results and the ultimate understanding of the technique.
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