A diagnostic test for scrapie-infected sheep using a capillary electrophoresis immunoassay with fluorescent-labeled peptides

Electrophoresis
Mary Jo Schmerr, A Jenny

Abstract

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...Continue Reading

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Feb 24, 2001·Electrophoresis·D SchmalzingC Piggee
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