The measurement of prion protein in bovine brain tissue using differentialextraction and DELFIA (R) as a diagnostic test for BSE

A simple diagnostic test for the detection of bovine spongiform encephalopa thy (BSE), based on a commercially available time-resolved fluorescence imm unoassay (DELFIA(R)) for the measurement of the normal and disease-associat ed isoforms of prion protein (PrP), is described. The isoforms are sequenti ally extracted from homogenized bovine brain tissue using two concentration s of guanidine hydrochloride. This procedure initially extracts a soluble i soform and subsequently a less soluble disease-associated aggregated isofor m. Following quantification of the two fractions, the percentage of the ins oluble prion becomes a measurable parameter, independent of protein concent ration, clearly identifying normal from infected animals displaying clinica l signs of BSE. The mean percentages of insoluble PrP in brain tissue from 60 BSE-confirmed-positive cattle and 100 cattle that had never been exposed to the disease were 52.6% (SD = 22.8) and 3.9% (SD = 1.5), respectively. T he assay is sensitive, with a detection limit of less than 50 pg PrP, and i s robust and precise (CVs < 10%) over the appropriate working range. Copyri ght (C) 2000 John Wiley & Sons, Ltd.