Comparison of detergent and protease enzyme combinations for the detection of scrapie-associated fibrils from the central nervous system of sheep naturally affected with scrapie.
Standardized samples of tissue from the central nervous system of four sheep naturally affected with scrapie and from four healthy control sheep were subjected to a centrifugal extraction technique used to obtain scrapie-associated fibrils; the latter were then demonstrated by negative-contrast transmission electron microscopy. This regime was used to evaluate the fibril yield obtained from the 25 possible combinations of five different detergents and five different proteolytic enzymes. N-lauroylsarcosine detergent was found to be the most efficient detergent for all five enzymes, followed by sulphabetaine 3-14. Sodium dodecyl sulphate detergent was successful only in combination with a subtilisin Carlsberg enzyme. Octylglucoside and nonidet P40 detergents did not produce fibrils with any of the enzymes. Proteinase K was the least efficient of the five enzymes when used in combination with N-lauroylsarcosine; subtilisin Carlsberg, clostripain, pronase and trypsin enzymes all gave higher fibril yields. A combination of N-lauroylsarcosine detergent and subtilisin Carlsberg proteolytic enzyme gave the highest fibril yield.
Stack
M J
MJ
Veterinary Laboratories Agency, Central Veterinary Laboratory, Weybridge, Surrey, UK.
Aldrich
A M
AM
Davis
L A
LA
D000818:Animals; D002490:Central Nervous System / Q000473:pathology*; D003902:Detergents; D004338:Drug Combinations; D010450:Endopeptidases; D005260:Female; D008854:Microscopy, Electron; D009454:Neurofibrils / Q000648:ultrastructure*; D012608:Scrapie / Q000473:pathology*; D012756:Sheep; D012757:Sheep Diseases / Q000473:pathology*
D003902:Detergents; D004338:Drug Combinations; D010450:Endopeptidases