SENSITIVE ENZYME-LINKED-IMMUNOSORBENT-ASSAY FOR DETECTION OF PRPSC INCRUDE TISSUE-EXTRACTS FROM SCRAPIE-AFFECTED MICE

An enzyme-linked immunosorbent assay (ELISA) was developed that detect s PrPSc in crude extracts from brain and spleen tissue of scrapie-affe cted mice with high sensitivity and specificity. Brain tissue was homo genized in 8% Zwittergent 3-12 and 0.5% Sarkosyl. The homogenate was t reated with collagenase and DNase I and then subjected to proteinase K digestion. Precipitates containing PrPSc were obtained by ultracentri fugation. Spleen tissue was homogenized in 4% Triton X-100 and 0.5% Sa rkosyl, and the homogenate was treated firstly with collagenase and DN ase I, and secondly with proteinase K. PrPSc was then extracted with 6 .25% Sarkosyl and precipitated through salting-out with NaCl and by ul tracentrifugation. When PrPSc was dissolved in 3-4 M guanidine thiocya nate and adsorbed to microtiter plates, strong and specific reactions to the formation of antigen-antibody complexes could be detected by EL ISA. The sensitivity of PrPSc-detection for this ELISA, as measured by serial dilution of scrapie material in tissue homogenates from uninfe cted animals, was equal or higher than that attained by Western blot. This ELISA is more rapid than Western blot and seems to be more suitab le for screening large numbers of animals. It also has potential appli cation for the diagnosis of the transmissible spongiform encephalopath ies. (C) 1997 Elsevier Science B.V.