Validation of a Western immunoblotting procedure for bovine PrP Sc detection and its use as a rapid surveillance method for the diagnosis of bovine spongiform encephalopathy (BSE)

Abstract

In this report we document the results of several independent studies testing the sensitivity, specificity and reliability of the Prionics Western blotting (PWB) procedure to detect bovine and ovine disease-specific, protease-resistant prion protein (PrP(Sc)). Validation of the technique was obtained by blind analysis of samples from cattle affected with bovine spongiform encephalopathy (BSE), clinically normal animals or cattle with neurological diseases unrelated to BSE. Overall, very high sensitivity, specificity and reliability was observed. It became clear that sampling of the correct brain region and the method used for protein extraction are important factors for correct diagnosis. Furthermore, we tested the usefulness of the PWB technique as an instrument for surveillance purposes. We analyzed animals from a culling scheme as well as older animals from abattoirs to determine the number of subclinical BSE cases detectable by histopathological examination, immunohistochemistry for PrP(Sc) and PWB. In both studies, BSE-affected animals with no overt clinical symptoms were detected. These results demonstrate the usefulness of the PWB procedure in surveillance systems serving as a rapid diagnostic tool to identify animals subclinically infected with BSE.

Degradation of scrapie associated prion protein (PrPSc) by the gastrointestinal microbiota of cattle

“…Transmissible spongiform encephalopathies (TSE) or "prion diseases" form a group of fatal neurodegenerative disorders, which include, among others, scrapie in sheep, bovine spongiform encephalopathy (BSE), and Creutzfeldt-Jakob disease leads to an N-terminally truncated form of 27-30 kDa, designated PrP Sc [15]. PrP Sc is resistant towards other proteolytic enzymes like trypsin, V8 protease [11] and pepsin [8].…”

Evaluation of Three Rapid Diagnostic Tests Used in Bovine Spongiform Encephalopathy Monitoring in Italy

“…In particular, the WB is based on a PrP-specific monoclonal antibody (6H4) c and an alkaline phosphataseconjugated antibody combined to a chemiluminescence detection system. 11 The sample is positive when the 3-band pattern of PrP27-30kD is identified. Each sample is tested in duplicate.…”

Effect of Time and Temperature on PrPCWD Immunoreactivity as Evidenced by Western Blot

“…However, it has largely been concluded that autolysis does not compromise the accuracy of assays that detect the proteinase-resistant, pathogenic isoform of the prion protein (PrP res ). 2,5,7,8,12,16 It was hypothesized that over a relatively long period of time, exposure to heat may have a more substantial effect on PrP res immunodection than previously considered. The objective of the present study was to characterize PrP CWD detectability with time in brain samples subjected to heat in vitro as determined by Western blot.…”