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A rapid dual staining procedure for the quantitative discrimination of prion amyloid from tissues reveals how interactions between amyloid and lipids in tissue homogenates may hinder the detection of prions

By R. Hervé, R. Collin, H.E. Pinchin, Thomas Secker and C.William Keevil

Abstract

Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative diseases with no cure to this day, and are often associated with the accumulation of amyloid plaques in the brain and other tissues in affected individuals. The emergence of new variant Creutzfeldt-Jakob disease, an acquired TSE with a relatively long asymptomatic incubation period and unknown prevalence or incidence, which could potentially be iatrogenically transmitted, has prompted the need for sensitive and rapid methods of detection of the pathology indicator, the protease-resistant prion protein (PrP(Sc)), in tissues and on surgical instruments. To discriminate between common tissue proteins and amyloid-rich aggregates such as those formed by abnormal prion, we developed a quantitative thioflavin T/SYPRO Ruby dual staining procedure, used in combination with episcopic differential interference contrast/epifluorescence (EDIC/EF) microscopy for rapid scanning of samples. The detection limit of this direct observation technique applied to brain homogenates was greatly enhanced by the addition of Tween 20, as demonstrated in double-blind studies using various proportions of ME7-infected brain mixed with normal brain homogenate. The characteristic thioflavin T signal correlated with the relative amount of prion amyloid and proved at least 2-log more sensitive than the classic Western blot using the same prepared samples. This new sensitive microscopy procedure, which can be easily applied in instrument decontamination surveys, is likely to be more sensitive that Western blot in practice since it does not rely on the elution of resilient PrP(Sc) bound to the instrument surfaces. Our study also demonstrates how interactions between prion and lipid-rich tissue homogenates may reduce the sensitivity of such detection assays

Topics: RA0421, RB, RC0321
Year: 2009
OAI identifier: oai:eprints.soton.ac.uk:157345
Provided by: e-Prints Soton

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  1. (2007). Amyloid adhesins are abundant in natural biofilms. doi
  2. (2008). Amyloid-like adhesins produced by floc-forming and filamentous bacteria in activated sludge. doi
  3. (2007). Amyloidspecific fluorophores for the rapid, sensitive in situ detection of prion contamination on surgical instruments. doi
  4. (1991). Bovine spongiform encephalopathy: a neuropathological perspective. doi
  5. (2006). Cell biology: infectious Alzheimer's disease? doi
  6. (2006). Clinical presentation and pre-mortem diagnosis of variant Creutzfeldt–Jakob disease associated with blood transfusion: a case report. doi
  7. (2008). Comparison between visual analysis and microscope assessment of surgical instrument cleanliness from sterile service departments. doi
  8. (2002). Debris on processed ophthalmic instruments: a cause for concern. doi
  9. et al.,1995. Western blot mapping of disease-specific amyloid in various animal species and humans with transmissible spongiform encephalopathies using a high-yield purification method. doi
  10. (2006). Exogenous induction of cerebral beta-amyloidogenesis is governed by agent and host. doi
  11. (2007). Formation of native prions from minimal components in vitro. doi
  12. H.,1993. Transmission of bovine spongiform encephalopathy to sheep and goats. doi
  13. (2007). Imaging beta-amyloid burden in aging and dementia. doi
  14. (2006). Kuru in the 21st century—an acquired human prion disease with very long incubation periods. doi
  15. (2007). Lipid interaction converts prion protein to a PrPSc-like proteinase K-resistant conformation under physiological conditions. doi
  16. (2004). Molecular aspects of disease pathogenesis in the transmissible spongiform encephalopathies. doi
  17. (2004). Possible transmission of variant Creutzfeldt–Jakob disease by blood transfusion. doi
  18. (2005). Prion diseases and iatrogenic infections I. A review.
  19. (2005). Prion diseases and iatrogenic infections II.
  20. (2001). Prion diseases of humans and animals: their causes and molecular basis. doi
  21. (2006). Prion diseases: current understanding of epidemiology and pathogenesis, and therapeutic advances. doi
  22. (2006). Prions in dentistry—what are they, should we be concerned, and what can we do?
  23. (2004). Protease-resistant human prion protein and ferritin are cotransported across Caco-2 epithelial cells: implications for species barrier in prion uptake from the intestine. doi
  24. (2003). Rapid detection of biofilms and adherent pathogens using scanning confocal laser microscopy and episcopic differential interference contrast microscopy.
  25. (2006). Rapid method for the sensitive detection of protein contamination on surgical instruments. doi
  26. (2005). Risk perception of the “mad cow disease” in France: determinants and consequences. doi
  27. (2001). Sensitive detection of pathological prion protein by cyclic amplification of protein misfolding. doi
  28. (2008). Serious Hazards of Transfusion Committee,
  29. (2003). Sporadic and familial CJD: classification and characterisation. doi
  30. (2004). Techniques to study amyloid fibril formation in vitro. doi
  31. (2006). The interaction between cytoplasmic prion protein and the hydrophobic lipid core of membrane correlates with neurotoxicity. doi
  32. (1998). The prion diseases. doi
  33. (1997). The same prion strain causes vCJD and BSE. doi
  34. (2006). The sensitivity of approved Ninhydrin and Biuret tests in the assessment of protein contamination on surgical steel as an aid to prevent iatrogenic prion transmission. doi
  35. (2003). The spectrum of safety: variant Creutzfeldt–Jakob disease in the United Kingdom. doi
  36. (2001). Tissue distribution of protease resistant prion protein in variant Creutzfeldt– Jakob disease using a highly sensitive immunoblotting assay. doi
  37. (2008). Transmissible spongiform encephalopathy in the 21st century: neuroscience for the clinical neurologist. doi
  38. (1996). Transmission dynamics and epidemiology of BSE in British cattle. doi
  39. (1997). Transmission of hepatitis C and prion diseases through digestive endoscopy: evaluation of risk and recommended practices. doi
  40. (2001). Variant Creutzfeldt–Jakob disease (vCJD) and gastrointestinal endoscopy. doi
  41. (2007). vCJD and dental treatment: where are we now? doi

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