Author
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Schmerr, Mary Jo |
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JENNY, ALLEN - USDA-APHIS-NVSL,AMES,IA |
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Submitted to: Electrophoresis
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 1/19/1998 Publication Date: N/A Citation: N/A Interpretive Summary: Scrapie is a naturally occurring disease in sheep and goats that is used as a model to study a group of diseases called transmissible spongiform encephalopathies. These diseases are found both in humans and animals and cause degeneration of the nervous system. There is no known treatment for this disease and the infected individuals die. The outbreak of bovine spongiform encephalopathy (Mad Cow Disease) in the United Kingdom is attributed to ingestion of scrapie contaminated bone meal. This has raised concern that a scrapie like disease may be transmitted to other domestic animals and humans through the food chain and through animal products used for cosmetics and medical purposes. It is important that tests are designed to detect this disease so that infected animals or contaminated materials can be removed to prevent transmission of this disease. The traditional tests for this disease are not sensitive enough to detect the minute amounts of material that may be present. Recently, new instrumentation using techniques that rely on the mobility of biological samples in an electric field has been developed. We used this instrumentation, called capillary electrophoresis, to develop a highly sensitive diagnostic test capable of detecting the scrapie agent in a minute amount (200ng) of brain sample. This diagnostic test will benefit the animal industries as well as those industries that produce pharmaceuticals and cosmetics from animal products. Technical 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 (PrP**sc) is a protease-resistant form of a normal host cell protein. When the aggregated protein is denatured in SDS Sand BETA-mercaptoethanol, a monomer form of Mr ca 27 kDa is observed. A competition immunoassay was developed using free zone capillary electrophoresis with LIF for detection and synthetic peptides from PrP**sc that were labeled with fluorescein to detect PrP**sc from scrapie infected sheep. Antibodies were made to each respective peptide. The fluorescent labeled peptides bound to the antibody were separated from the unbound peptides using 200 mM Tricine pH 8.0 containing 0.1percent N-octylglucoside and 0.1percent BSA. The amount of antibody that would bind ca 50 percent of fthe fluorescent labeled peptide was determined for each peptide used. When unlabeled peptide was added to the assay, ca 2 fmoles of the peptide could be measured. When PrP**sc extracted from infected sheep brain was added to the assay, approximately 135 pg of PrP**sc 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. |
