Detection of prion particles in samples of BSE and scrapie by fluorescence correlation spectroscopy without proteinase K digestion

Birkmann, Eva; Schäfer, Oliver; Weinmann, Nicole; Dumpitak, Christian; Beekes, Michael; Jackman, R.; Thorne, Leigh; Riesner, Detlev

doi:10.1515/bc.2006.013

Cited by 34 publications

(31 citation statements)

References 23 publications

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“…Our ASA is able to detect protease-sensitive prions more readily than the CDI. Other techniques have been reported for the detection of sPrP Sc after PTA precipitation but they are not currently in routine use and not as simple to apply as the ASA (25,26). Bioassays, although extremely sensitive, require long incubation times (60-300 days) to yield results and, thus, are costly.…”

Section: Discussionmentioning

confidence: 99%

Prion detection by an amyloid seeding assay

Colby

Zhang²,

Wang³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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207

185

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Polymerization of recombinant prion protein (recPrP), which was produced in bacteria, into amyloid fibers was accompanied by the acquisition of prion infectivity. We report here that partially purified preparations of prions seed the polymerization of recPrP into amyloid as detected by a fluorescence shift in the dye Thioflavin T. Our amyloid seeding assay (ASA) detected PrP Sc , the sole component of the prion, in brain samples from humans with sporadic Creutzfeldt–Jakob disease, as well as in rodents with experimental prion disease. The ASA detected a variety of prion strains passaged in both mice and hamsters. The sensitivity of the ASA varied with strain type; for hamster Sc237 prions, the limit of detection was ≈1 fg. Some prion strains consist largely of protease-sensitive PrP Sc (sPrP Sc ), and these strains were readily detected by ASA. Our studies show that the ASA provides an alternative methodology for detecting both sPrP Sc and protease-resistant PrP Sc that does not rely on protease digestion or immunodetection.

show abstract

Section: Discussionmentioning

confidence: 99%

Prion detection by an amyloid seeding assay

Colby

Zhang²,

Wang³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

207

185

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show abstract

“…The seeds, however, were naturally occurring PrP Sc purified by PTA precipitation (22). Although we do not claim that no cellular factors are involved in prion infection, they are currently unknown and cannot be taken into account.…”

Section: Discussionmentioning

confidence: 88%

“…We analyzed the effect of PrP Sc seeds on the mechanism and kinetics of fibril formation of recPrP. PrP Sc seeds were prepared from the brain of an infected hamster by NaPTA precipitation (22), and negative controls were obtained in the same manner from noninfected brain tissue. To evaluate the kinetics quantitatively, we applied a homogeneous system, without the cyclic sonication of the PMCA system (15), but with thorough shaking.…”

Section: Resultsmentioning

confidence: 99%

“…Perhaps a prion amplification system of intermediate sensitivity can be combined with a detection system of robust sensitivity. Alternatively, we have applied single-prion particle counting by surface-FIDA for diagnostic purposes (22,34), which might be improved in the future by combination with an amplification system as described here.…”

Section: Discussionmentioning

confidence: 99%

“…It was expressed, purified, and refolded as described (25,35). PrP Sc was isolated to high purity by using a modified protocol of the NaPTA precipitation method (22,36). Differing from the protocol, an additional washing step in 10 mM NaPi was performed and the resulting pellet was resuspended in 10 mM NaPi by brief sonication.…”

Section: Methodsmentioning

confidence: 99%

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Mechanisms of prion protein assembly into amyloid

Stöhr

Weinmann

Wille

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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130

112

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The conversion of the ␣-helical, cellular isoform of the prion protein (PrP C ) to the insoluble, ␤-sheet-rich, infectious, diseasecausing isoform (PrP Sc ) is the key event in prion diseases. In an earlier study, several forms of PrP were converted into a fibrillar state by using an in vitro conversion system consisting of low concentrations of SDS and 250 mM NaCl. Here, we characterize the structure of the fibril precursor state, that is, the soluble state under fibrillization conditions. CD spectroscopy, analytical ultracentrifugation, and chemical cross-linking indicate that the precursor state exists in a monomer-dimer equilibrium of partially denatured, ␣-helical PrP, with a well defined contact site of the subunits in the dimer. Using fluorescence with thioflavin T, we monitored and quantitatively described the kinetics of seeded fibril formation, including dependence of the reaction on substrate and seed concentrations. Exponential, seed-enhanced growth can be achieved in homogeneous solution, which can be enhanced by sonication. From these data, we propose a mechanistic model of fibrillization, including the presence of several intermediate structures. These studies also provide a simplified amplification system for prions.dimer ͉ seeding ͉ fibril ͉ precursor state

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Capillary electromigration based techniques in diagnostics of prion protein caused diseases

et al. 2012

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Transmissible spongiform encephalopathies 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 above-mentioned 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 abnormal prion protein (PrP(Sc) ) detection. On the other hand, using of CE 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 CE, CIEF, and/or CGE. The potential of the miniaturized and integrated lab-on-chip devices is highlighted, emphasizing recent advances of this field in the proteomic analysis.

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Detection of prion particles in samples of BSE and scrapie by fluorescence correlation spectroscopy without proteinase K digestion

Cited by 34 publications

References 23 publications

Prion detection by an amyloid seeding assay

Prion detection by an amyloid seeding assay

Mechanisms of prion protein assembly into amyloid

Capillary electromigration based techniques in diagnostics of prion protein caused diseases

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