The measurement of prion protein in bovine brain tissue using differential extraction and DELFIA as a diagnostic test for BSE.
A simple diagnostic test for the detection of bovine spongiform encephalopathy (BSE), based on a commercially available time-resolved fluorescence immunoassay (DELFIA) for the measurement of the normal and disease-associated isoforms of prion protein (PrP), is described. The isoforms are sequentially extracted from homogenized bovine brain tissue using two concentrations of guanidine hydrochloride. This procedure initially extracts a soluble isoform and subsequently a less soluble disease-associated aggregated isoform. Following quantification of the two fractions, the percentage of the insoluble prion becomes a measurable parameter, independent of protein concentration, clearly identifying normal from infected animals displaying clinical 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. The assay is sensitive, with a detection limit of less than 50 pg PrP, and is robust and precise (CVs < 10%) over the appropriate working range.
Barnard
G
G
Endocrine Unit, Department of Chemical Pathology, Level D, South Pathology Block, Southampton General Hospital, Tremona Road, Southampton SO16 6YD, UK. GJRB2@aol.com
Helmick
B
B
Madden
S
S
Gilbourne
C
C
Patel
R
R
D000818:Animals; D001923:Brain Chemistry; D002417:Cattle; D016643:Encephalopathy, Bovine Spongiform / Q000175:diagnosis*; D019286:Endopeptidase K; D015200:Fluoroimmunoassay / Q000379:methods / Q000592:standards / Q000662:veterinary*; D011328:Prions / Q000032:analysis* / Q000302:isolation & purification; D012995:Solubility
D011328:Prions; D019286:Endopeptidase K