A diagnostic test for scrapie-infected sheep using a capillary electrophoresis immunoassay with fluorescent-labeled peptides.
Scrapie in sheep and goats is the prototype of transmissible spongiform encephalopathies found in humans and animals. A feature of these diseases is the accumulation of rod-shaped fibrils in the brain that form from an aggregated protein. This protein (PrPSC) is a protease-resistant form of a normal host cell protein. When the aggregated protein is denatured in sodium dodecyl sulfate (SDS) and beta-mercaptoethanol, a monomer form of approximately 27 kDa molecular mass is observed. A competition immunoassay to detect PrPSC from scrapie-infected sheep was developed using free zone capillary electrophoresis with laser-induced fluorescence (LIF) for detection and flourescein-labeled synthetic peptides from PrPSC. Antibodies were made to each respective peptide and used in the competition assay. The fluorescent-labeled peptides bound to the antibody were separated from the unbound peptides using 200 mM Tricine, pH 8.0, containing 0.1% n-octylglucoside and 0.1% bovine serum albumin (BSA). The amount of antibody that would bind approximately 50% of the fluorescent-labeled peptide was determined for each peptide. When unlabeled peptide was added to the assay, approximately 2 fmoles of the peptide could be measured. When PrPSC extracted from infected sheep brain was added to the assay, approximately 135 pg of PrPSC could be detected. When preparations from normal sheep were assayed, there was little or no competition for the bound peptides. Assays using two of the peptides, peptides spanning amino acid positions 142-154 and 155-178, clearly differentiated scrapie-positive sheep from normal animals. This assay is a new method that can be used to diagnose scrapie and, possibly, other transmissible spongiform encephalopathies in animals and in humans.
Schmerr
M J
MJ
National Animal Disease Center, US Department of Agriculture, Agricultural Research Service, Ames, IA 50010, USA.
Jenny
A
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D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D019075:Electrophoresis, Capillary / Q000379:methods* / Q000706:statistics & numerical data; D019793:Fluorescein; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D018620:PrPSc Proteins / Q000737:chemistry; D015203:Reproducibility of Results; D012608:Scrapie / Q000175:diagnosis*; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine; D012756:Sheep
D010446:Peptide Fragments; D018620:PrPSc Proteins; D012710:Serum Albumin, Bovine; D019793:Fluorescein