NR AKNO
AU Schmerr,M.J.; Goodwin,K.R.; Cutlip,R.C.
TI Capillary electrophoresis of the scrapie prion protein from sheep brain
QU Journal of Chromatography. A 1994 Oct 7; 680(2): 447-53
PT journal article
AB Scrapie in sheep and goats causes a progressive, degenerative disease of the central nervous system and is the prototype of other transmissible spongiform encephalopathies (TSE) found in humans and in animals. In samples of TSE-affected brains, unique rod-shaped structures are found and are infectious. These rods are composed of a protease-resistant, post-translationally modified cellular protein (PrPsc) that has a molecular mass of ca. 27,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Laboratory tests used for the diagnosis of scrapie detect PrPsc. The overall concentration of PrPsc in tissues is low. The present methods to diagnose scrapie are lengthy, require relatively large quantities of starting material to detect PrPsc and lack sensitivity. We explored the use of free zone capillary electrophoresis and immunocomplex formation to detect PrPsc in the brain tissue of infected sheep. Brain tissue from both infected (as confirmed by histological and biological tests) and from normal animals was used to prepare the PrPsc. After treatment with proteinase K and non-ionic detergents, PrPsc was solubilized and reacted with a rabbit antiserum specific for a peptide of the prion protein. Immunocomplex formation was observed for the samples from scrapie-infected brain but not for samples from normal brain. When a fluorescein-labeled goat anti-rabbit immunoglobulin was used as a second antibody, the detection of immunocomplex formation was enhanced both by the immunological technique and by using laser-induced fluorescence for detection. This same rabbit antiserum was used on immunoblot analysis. Three bands were observed for material from an infected sheep but none in preparations from brain material from normal sheep. Capillary electrophoresis can be used to show immunocomplex formation when PrPsc is present in sheep brain.
IN Aus Gehirnmaterial normaler bzw. scrapieinfizierter Schafe wurden Proteine isoliert, mit Proteinase K verdaut und mit nichtionischen Detergentien gelöst. Nur wenn das Gehirnmaterial von einem scrapieinfizierten Schaf stammte, wurde die Bildung von Immunkomplexen mit Kaninchenantiserum gegen Prionprotein beobachtet. Mit dem gleichen Kaninchenantiserum wurden auf Immunoblots 3 scrapiespezifische Banden markiert. Auch mittels Kapillarelektrophorese konnten die Immunkomplexformation nachgewiesen werden.
ZR 19
MH Animal; *Brain Chemistry; Capillarity; Detergents; Electrophoresis/*methods; Electrophoresis, Polyacrylamide Gel; Endopeptidase K; Fluorescence; Fluorescent Antibody Technique; Immunoblotting; Lasers; Molecular Weight; Prions/*analysis; Scrapie/*diagnosis; Serine Endopeptidases; Sheep
AD National Animal Disease Center, US Department of Agriculture, Ames, IA 50010.
SP englisch
PO Niederlande