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ARS Home » Research » Publications at this Location » Publication #66120
Title: IMPROVEMENTS IN A COMPETITION ASSAY TO DETECT SCRAPIE PRION PROTEIN BY CAPILLARY ELECTROPHORESIS.

Author
item Schmerr, Mary Jo
item Goodwin, Kathryn
item Cutlip, Randall
item JENNY, ALLEN - PL,NVSL,AMES,IA

Submitted to: Journal of Chromatography
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/25/1996
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 for possible transmission 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 prevent this possible transmission from contaminated materials. Since present tests are not able to detect minute amounts of material that may be present in this samples, a more sensitive test is needed to eliminate this disease in animals. Recently, a new technique based on the mobility of biological samples in an electric field has been developed. We used this technique, called capillary electrophoresis, to detect the presence of this disease agent at 1 part per billion. This test has the potential to be used as a diagnostic test to identify the presence of the disease agent in contaminated samples.

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 is a protease-resistant form of a normal host cell protein. When the aggregated protein is denatured in SDS and beta-mercaptoethanol, a monomeric form (prion protein) of molecular mass of 27 Kdaltons is observed. Free zone capillary electrophoresis and peptides labeled with fluorescein iodacetamide were used to detect the prion protein through competition for a labeled peptide in immunocomplex formation. The separation of the immunocomplexes from the unbound peptide with the buffer 200mM Tricine pH 8.0 was faster and was better resolved than that obtained with phosphate or borate buffer systems. The amount of immunocomplex formation was dependent on the amount of antibody in the assay. The amount of bound labeled peptide could be measured directly as could the amount of unbound labeled peptide. As increasing amounts of unlabeled peptide were added to the assay, a concentration dependent reduction in the immunocomplex peak was observed. The assay could detect less than 10.0fmole of unlabeled peptide. There was a quantitative difference in the competition of preparations from scrapie infected sheep brain and normal sheep brain.