Western blot mapping of disease-specific amyloid in various animal species and humans with transmissible spongiform encephalopathies using a high-yield purification method.
SAF-protein, an amyloid, is the main constituent of scrapie-associated fibrils (SAF) and a specific marker for transmissible spongiform encephalopathies (TSE). Using an improved extraction method and Western blot detection, the disease-specific amyloid was found in various parts of the central nervous system of hamsters orally infected with scrapie, of squirrel monkeys orally infected with kuru, sporadic Creutzfeldt-Jakob disease (CJD) and scrapie, of human patients with sporadic CJD, of a sheep with natural scrapie and of a cow with bovine spongiform encephalopathy (BSE). In human CJD samples, the concentration of TSE-specific amyloid was estimated to be 1000- to 10 000-fold lower than in the central nervous system of hamsters with scrapie. The extraction method has a yield of 70% and allows Western blot detection of the TSE-specific amyloid in samples representing 1-10 micrograms of brain tissue from intracerebrally infected hamsters, as well as in individual spleens from hamsters with terminal scrapie infected by the intracerebral, oral or intraperitoneal route. A 20-100 mg sample of material is sufficient for the extraction of the pathological protein from different rodent, monkey, ovine, bovine and human tissues. The results reported here demonstrate the potential suitability of the method for the routine diagnosis of TSE as well as for the detailed analysis of distribution patterns of the TSE-specific amyloid in experimental approaches to the investigation of these diseases.
Beekes
M
M
Robert Koch-Institut, Bundesinstitut für Infektionskrankheiten und nicht übertragbare Krankheiten, Berlin, Germany.
Baldauf
E
E
Cassens
S
S
Diringer
H
H
Keyes
P
P
Scott
A C
AC
Wells
G A
GA
Brown
P
P
Gibbs
C J
CJ
Gajdusek
D C
DC
D000682:Amyloid / Q000302:isolation & purification*; D000818:Animals; D015153:Blotting, Western / Q000379:methods*; D002417:Cattle; D002490:Central Nervous System / Q000737:chemistry*; D006224:Cricetinae; D005260:Female; D006801:Humans; D008647:Mesocricetus; D017096:Prion Diseases / Q000175:diagnosis / Q000378:metabolism* / Q000662:veterinary; D011498:Protein Precursors / Q000032:analysis; D011506:Proteins / Q000032:analysis; D012453:Saimiri; D012756:Sheep; D013154:Spleen / Q000737:chemistry
D000682:Amyloid; D011498:Protein Precursors; D011506:Proteins; C056174:amyloid fibril protein SAF