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.
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.
Hervé
R
R
Environmental Healthcare Unit, School of Biological Sciences, University of Southampton, Southampton, UK. R.Herve@soton.ac.uk
Collin
R
R
Pinchin
H E
HE
Secker
T
T
Keevil
C W
CW