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Sensitive and specific detection of sporadic Creutzfeldt-Jakob disease brain prion protein using real-time quaking-induced conversion.

ABSTRACT: Real-time quaking-induced conversion (RT-QuIC) is an assay in which disease-associated prion protein (PrP) initiates a rapid conformational transition in recombinant PrP (recPrP), resulting in the formation of amyloid that can be monitored in real time using the dye thioflavin T. It therefore has potential advantages over analogous cell-free PrP conversion assays such as protein misfolding cyclic amplification (PMCA). The QuIC assay and the related amyloid seeding assay have been developed largely using rodent-passaged sheep scrapie strains. Given the potential RT-QuIC has for Creutzfeldt-Jakob disease (CJD) research and human prion test development, this study characterized the behaviour of a range of CJD brain specimens with hamster and human recPrP in the RT-QuIC assay. The results showed that RT-QuIC is a rapid, sensitive and specific test for the form of abnormal PrP found in the most commonly occurring forms of sporadic CJD. The assay appeared to be largely independent of species-related sequence differences between human and hamster recPrP and of the methionine/valine polymorphism at codon 129 of the human PrP gene. However, with the same conditions and substrate, the assay was less efficient in detecting the abnormal PrP that characterizes variant CJD brain. Comparison of these QuIC results with those previously obtained using PMCA suggested that these two seemingly similar assays differ in important respects.

SUBMITTER: Peden AH

PROVIDER: S-EPMC3352348 | biostudies-literature | 2012 Feb

REPOSITORIES: biostudies-literature

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Sensitive and specific detection of sporadic Creutzfeldt-Jakob disease brain prion protein using real-time quaking-induced conversion.

Peden Alexander H AH McGuire Lynne I LI Appleford Nigel E J NEJ Mallinson Gary G Wilham Jason M JM Orrú Christina D CD Caughey Byron B Ironside James W JW Knight Richard S RS Will Robert G RG Green Alison J E AJE Head Mark W MW

The Journal of general virology 20111026 Pt 2

Real-time quaking-induced conversion (RT-QuIC) is an assay in which disease-associated prion protein (PrP) initiates a rapid conformational transition in recombinant PrP (recPrP), resulting in the formation of amyloid that can be monitored in real time using the dye thioflavin T. It therefore has potential advantages over analogous cell-free PrP conversion assays such as protein misfolding cyclic amplification (PMCA). The QuIC assay and the related amyloid seeding assay have been developed large ...[more]

PMID: 22031526

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Project description:ImportanceCreutzfeldt-Jakob disease (CJD) is a fatal neurodegenerative disorder associated with the accumulation of infectious abnormal prion protein through a mechanism of templated misfolding. A recent report has described the detection of abnormal prion protein in the urine of patients with variant CJD (vCJD) using protein misfolding by cyclic amplification, which was apparently absent in the more common sporadic form of CJD (sCJD). A noninvasive diagnostic test could improve early diagnosis of sCJD and, by screening donations, mitigate the potential risks of prion transmission through human urine-derived pharmaceuticals. Here, we describe the adaptation of the direct detection assay, developed originally as a blood test for vCJD, for the detection of disease-associated prion protein in urine samples from patients with sCJD.ObjectiveTo determine the feasibility of sCJD diagnosis by adaptation of an established vCJD diagnostic blood test to urine.Design, setting, and participantsThis retrospective, cross-sectional study included anonymized urine samples from healthy nonneurological control individuals (n = 91), patients with non-prion neurodegenerative diseases (n = 34), and patients with prion disease (n = 37) of which 20 had sCJD. Urine samples obtained during the Medical Research Council PRION-1 Trial, the National Prion Monitoring Cohort Study, and/or referred to the National Prion Clinic or Dementia Research Centre at the National Hospital for Neurology and Neurosurgery in the United Kingdom.Main outcomes and measuresPresence of sCJD infection determined by an assay that captures, enriches, and detects disease-associated prion protein isoforms.ResultsA total of 162 samples were analyzed, composed of 91 normal control individuals (51 male, 33 female, and 7 not recorded), 34 neurological disease control individuals (19 male and 15 female), and 37 with prion disease (22 male and 15 female). The assay's specificity for prion disease was 100% (95% CI, 97%-100%), with no false-positive reactions from 125 control individuals, including 34 from a range of neurodegenerative diseases. In contrast to a previous study, which used a different method, sensitivity to vCJD infection was low (7.7%; 95% CI, 0.2%-36%), with only 1 of 13 patients with positive test results, while sensitivity to sCJD was unexpectedly high at 40% (95% CI, 19%-64%).Conclusions and relevanceWe determined 40% of sCJD urine sample results as positive. To our knowledge, this is the first demonstration of an assay that can detect sCJD infection in urine or any target analyte outside of the central nervous system. Urine detection could allow the development of rapid, molecular diagnostics for sCJD and has implications for other neurodegenerative diseases where disease-related assemblies of misfolded proteins might also be present in urine.

Project description:BackgroundPrion diseases are a group of invariably fatal neurodegenerative disorders affecting humans and a wide range of mammals. An essential part of the infectious agent, termed the prion, is composed of an abnormal isoform (PrPSc) of a host-encoded normal cellular protein (PrPC). The conversion of PrPC to PrPSc is thought to play a crucial role in the development of prion diseases and leads to PrPSc deposition, mainly in the central nervous system. Sporadic Creutzfeldt-Jakob disease (sCJD), the most common form of human prion disease, presents with a marked clinical heterogeneity. This diversity is accompanied by a molecular signature which can be defined by histological, biochemical, and genetic means. The molecular classification of sCJD is an important tool to aid in the understanding of underlying disease mechanisms and the development of therapy protocols. Comparability of classifications is hampered by disparity of applied methods and inter-observer variability.Methods and findingsTo overcome these difficulties, we developed a new quantification protocol for PrPSc by using internal standards on each Western blot, which allows for generation and direct comparison of individual PrPSc profiles. By studying PrPSc profiles and PrPSc type expression within nine defined central nervous system areas of 50 patients with sCJD, we were able to show distinct PrPSc distribution patterns in diverse subtypes of sCJD. Furthermore, we were able to demonstrate the co-existence of more than one PrPSc type in individuals with sCJD in about 20% of all patients and in more than 50% of patients heterozygous for a polymorphism on codon 129 of the gene encoding the prion protein (PRNP).ConclusionPrPSc profiling represents a valuable tool for the molecular classification of human prion diseases and has important implications for their diagnosis by brain biopsy. Our results show that the co-existence of more than one PrPSc type might be influenced by genetic and brain region-specific determinants. These findings provide valuable insights into the generation of distinct PrPSc types.

Dataset Information

Sensitive and specific detection of sporadic Creutzfeldt-Jakob disease brain prion protein using real-time quaking-induced conversion.

Publications

Sensitive and specific detection of sporadic Creutzfeldt-Jakob disease brain prion protein using real-time quaking-induced conversion.

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