Detection of bovine spongiform encephalopathy-specific PrP(Sc) by treatment with heat and guanidine thiocyanate.
The conversion of a ubiquitous cellular protein (PrP(C)), an isoform of the prion protein (PrP), to the pathology-associated isoform PrP(Sc) is one of the hallmarks of transmissible spongiform encephalopathies such as bovine spongiform encephalopathy (BSE). Accumulation of PrP(Sc) has been used to diagnose BSE. Here we describe a quantitative enzyme-linked immunosorbent assay (ELISA) that involves antibodies against epitopes within the protease-resistant core of the PrP molecule to measure the amount of PrP in brain tissues from animals with BSE and normal controls. In native tissue preparations, little difference was found between the two groups. However, following treatment of the tissue with heat and guanidine thiocyanate (Gh treatment), the ELISA discriminated BSE-specific PrP(Sc) from PrP(C) in bovine brain homogenates. PrP(Sc) was identified by Western blot, centrifugation, and protease digestion experiments. It was thought that folding or complexing of PrP(Sc) is most probably reversed by the Gh treatment, making hidden antigenic sites accessible. The digestion experiments also showed that protease-resistant PrP in BSE is more difficult to detect than that in hamster scrapie. While the concentration of PrP(C) in cattle is similar to that in hamsters, PrP(Sc) sparse in comparison. The detection of PrP(Sc) by a simple physicochemical treatment without the need for protease digestion, as described in this study, could be applied to develop a diagnostic assay to screen large numbers of samples.
Meyer
R K
RK
BSE Reference Center, Institute of Animal Neurology, University of Bern, CH-3012 Bern, Switzerland. meyer@itn.unibe.ch
Oesch
B
B
Fatzer
R
R
Zurbriggen
A
A
Vandevelde
M
M
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000914:Antibodies, Viral / Q000276:immunology; D015153:Blotting, Western; D001921:Brain / Q000473:pathology; D001923:Brain Chemistry; D002417:Cattle; D002540:Cerebral Cortex / Q000737:chemistry; D006224:Cricetinae; D016643:Encephalopathy, Bovine Spongiform / Q000175:diagnosis*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006146:Guanidines; D006358:Hot Temperature; D008526:Medulla Oblongata / Q000737:chemistry; D018621:PrPC Proteins / Q000032:analysis; D018620:PrPSc Proteins / Q000032:analysis*; D013788:Thalamus / Q000737:chemistry; D013861:Thiocyanates; D014020:Tissue Extracts
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D006146:Guanidines; D018621:PrPC Proteins; D018620:PrPSc Proteins; D013861:Thiocyanates; D014020:Tissue Extracts; C054436:guanidine thiocyanate