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.