Sensitive enzyme-linked immunosorbent assay for detection of PrP(Sc) in crude tissue extracts from scrapie-affected mice.
An enzyme-linked immunosorbent assay (ELISA) was developed that detects PrP(Sc) in crude extracts from brain and spleen tissue of scrapie-affected mice with high sensitivity and specificity. Brain tissue was homogenized in 8% Zwittergent 3-12 and 0.5% Sarkosyl. The homogenate was treated with collagenase and DNase I and then subjected to proteinase K digestion. Precipitates containing PrP(Sc) were obtained by ultracentrifugation. Spleen tissue was homogenized in 4% Triton X-100 and 0.5% Sarkosyl, and the homogenate was treated firstly with collagenase and DNase I, and secondly with proteinase K. PrP(Sc) was then extracted with 6.25% Sarkosyl and precipitated through salting-out with NaCl and by ultracentrifugation. When PrP(Sc) was dissolved in 3-4 M guanidine thiocyanate and adsorbed to microtiter plates, strong and specific reactions to the formation of antigen-antibody complexes could be detected by ELISA. The sensitivity of PrP(Sc)-detection for this ELISA, as measured by serial dilution of scrapie material in tissue homogenates from uninfected animals, was equal or higher than that attained by Western blot. This ELISA is more rapid than Western blot and seems to be more suitable for screening large numbers of animals. It also has potential application for the diagnosis of the transmissible spongiform encephalopathies.
Grathwohl
K U
KU
Department of Veterinary Public Health, Obihiro University of Agriculture and Veterinary Medicine, Hokkaido, Japan.
Horiuchi
M
M
Ishiguro
N
N
Shinagawa
M
M
D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000473:pathology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D051379:Mice; D008813:Mice, Inbred ICR; D018620:PrPSc Proteins / Q000032:analysis*; D012608:Scrapie / Q000378:metabolism* / Q000473:pathology; D012680:Sensitivity and Specificity; D013154:Spleen / Q000473:pathology; D014020:Tissue Extracts
D018620:PrPSc Proteins; D014020:Tissue Extracts