Ultra-efficient replication of infectious prions by automated protein misfolding cyclic amplification.
ABSTRACT Prions are the unconventional infectious agents responsible for transmissible spongiform encephalopathies, which appear to be composed mainly or exclusively of the misfolded prion protein (PrPSc). Prion replication involves the conversion of the normal prion protein (PrPC) into the misfolded isoform, catalyzed by tiny quantities of PrPSc present in the infectious material. We have recently developed the protein misfolding cyclic amplification (PMCA) technology to sustain the autocatalytic replication of infectious prions in vitro. Here we show that PMCA enables the specific and reproducible amplification of exceptionally minute quantities of PrPSc. Indeed, after seven rounds of PMCA, we were able to generate large amounts of PrPSc starting from a 1x10(-12) dilution of scrapie hamster brain, which contains the equivalent of approximately 26 molecules of protein monomers. According to recent data, this quantity is similar to the minimum number of molecules present in a single particle of infectious PrPSc, indicating that PMCA may enable detection of as little as one oligomeric PrPSc infectious particle. Interestingly, the in vitro generated PrPSc was infectious when injected in wild-type hamsters, producing a disease identical to the one generated by inoculation of the brain infectious material. The unprecedented amplification efficiency of PMCA leads to a several billion-fold increase of sensitivity for PrPSc detection as compared with standard tests used to screen prion-infected cattle and at least 4000 times more sensitivity than the animal bioassay. Therefore, PMCA offers great promise for the development of highly sensitive, specific, and early diagnosis of transmissible spongiform encephalopathy and to further understand the molecular basis of prion propagation.
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ABSTRACT: Transmissible spongiform encephalopathies (TSEs) are a group of fatal neurodegenerative diseases with long incubation time. This group includes Creutzfeld-Jakob disease, kuru, scrapie, chronic wasting disease, and bovine spongiform encephalopathy. Sensitive and specific detection of abnormal prion protein as "a source agent" of the abovementioned diseases in blood could provide a diagnostic test or a screening assay for animal and human prion protein diseases diagnostics. Therefore, diagnostic tests for prion protein diseases represent unique challenge requiring development of novel assays exploiting properties of prion protein complex. Presently, diagnostic methods such as protein misfolding cyclic amplification, conformation-dependent immunoassay, dissociation-enhanced lanthanide fluorescent immunoassay, fluorescence correlation spectroscopy, and/or flow microbead immunoassay are used for PrP(Sc) detection. On the other hand, using of capillary electrophoresis for PrP(Sc) detection in body fluids is an attractive alternative; it has been already applied for the blood samples of infected sheep, elk, chimpanzee as well as humans. In this review, assays for prion protein detection are summarized with special attention to capillary electromigration based techniques, such as capillary electrophoresis, capillary isoelectric focusing, and/or capillary gel electrophoresis. The potential of the miniaturized and integrated lab-on-chip devices is highlighted, emphasizing recent advances of this field in the proteomic analysis.Electrophoresis 09/2012; · 3.26 Impact Factor
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ABSTRACT: Prion diseases are fatal neurodegenerative disorders characterized by misfolding of the cellular prion protein (PrP(c)) into the disease-associated isoform (PrP(Sc)) that has increased β-sheet content and partial resistance to proteolytic digestion. Prion diseases from different mammalian species have varying propensities for transmission upon exposure of an uninfected host to the infectious agent. Chronic Wasting Disease (CWD) is a highly transmissible prion disease that affects free ranging and farmed populations of cervids including deer, elk and moose, as well as other mammals in experimental settings. The molecular mechanisms allowing CWD to maintain comparatively high transmission rates have not been determined. Previous work has identified a unique structural feature in cervid PrP, a rigid loop between β-sheet 2 and α-helix 2 on the surface of the protein. This study was designed to test the hypothesis that the rigid loop has a direct influence on the misfolding process. The rigid loop was introduced into murine PrP as the result of two amino acid substitutions: S170N and N174T. Wild-type and rigid loop murine PrP were expressed in E. coli and purified. Misfolding propensity was compared for the two proteins using biochemical techniques and cell free misfolding and conversion systems. Murine PrP with a rigid loop misfolded in cell free systems with greater propensity than wild type murine PrP. In a lipid-based conversion assay, rigid loop PrP converted to a PK resistant, aggregated isoform at lower concentrations than wild-type PrP. Using both proteins as substrates in real time quaking-induced conversion, rigid loop PrP adopted a misfolded isoform more readily than wild type PrP. Taken together, these findings may help explain the high transmission rates observed for CWD within cervids.PLoS ONE 01/2013; 8(6):e66715. · 3.73 Impact Factor
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ABSTRACT: Variant Creutzfeldt-Jakob disease (vCJD) is a neurodegenerative infectious disorder, characterized by a prominent accumulation of pathological isoforms of the prion protein (PrP(TSE)) in the brain and lymphoid tissues. Since the publication in the United Kingdom of four apparent vCJD cases following transfusion of red blood cells and one apparent case following treatment with factor VIII, the presence of vCJD infectivity in the blood seems highly probable. For effective blood testing of vCJD individuals in the preclinical or clinical phase of infection, it is considered necessary that assays detect PrP(TSE) concentrations in the femtomolar range. We have developed a three-step assay that firstly captures PrP(TSE) from infected blood using a plasminogen-coated magnetic-nanobead method prior to its serial amplification via protein misfolding cyclic amplification (PMCA) and specific PrP(TSE) detection by western blot. We achieved a PrP(TSE) capture yield of 95% from scrapie-infected material. We demonstrated the possibility of detecting PrP(TSE) in white blood cells, in buffy coat and in plasma isolated from the blood of scrapie-infected sheep collected at the pre-clinical stage of the disease. The test also allowed the detection of PrP(TSE) in human plasma spiked with a 10(-8) dilution of vCJD-infected brain homogenate corresponding to the level of sensitivity (femtogram) required for the detection of the PrP(TSE) in asymptomatic carriers. The 100% specificity of the test was revealed using a blinded panel comprising 96 human plasma samples. We have developed a sensitive and specific amplification assay allowing the detection of PrP(TSE) in the plasma and buffy coat fractions of blood collected at the pre-clinical phase of the disease. This assay represents a good candidate as a confirmatory assay for the presence of PrP(TSE) in blood of patients displaying positivity in large scale screening tests.PLoS ONE 01/2013; 8(7):e69632. · 3.73 Impact Factor