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 spongifor m encephalopathies found in humans and animals. A feature of these dis eases is the accumulation of rod-shaped fibrils in the brain that form from an aggregated protein. This protein (PrPSC) is a protease-resist ant 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 similar to 27 kDa molecular mass is observed. A compe tition immunoassay to detect PrPSC from scrapie-infected sheep was dev eloped using free zone capillary electrophoresis with laser-induced fl uorescence (LIF) for detection and flourescein-labeled synthetic pepti des from PrPSC. Antibodies were made to each respective pep tide and u sed in the competition assay. The fluorescent-labeled peptides bound t o the antibody were separated from the unbound peptides using 200 mM T ricine, pH 8.0, containing 0.1% n-octylglucoside and 0.1% bovine serum albumin (BSA). The amount of antibody that would bind similar to 50% of the fluorescent-labeled peptide was determined for each peptide. Wh en unlabeled peptide was added to the assay, similar to 2 fmoles of th e peptide could be measured. When PrPSC extracted from infected sheep brain was added to the assay, approximately 135 pg of PrPSC could be d etected. When preparations from normal sheep were assayed, there was l ittle or no competition for the bound peptides. Assays using two of th e peptides, peptides spanning amino acid positions 142-154 and 155-178 , clearly differentiated scrapie-positive sheep from normal animals. T his assay is a new method that can be used to diagnose scrapie and, po ssibly, other transmissible spongiform encephalopathies in animals and in humans.