|Schmerr, Mary Jo|
Submitted to: Journal of Chromatography
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/19/1998
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 these 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 detect the presence of this disease agent in approximately 50 ug of brain sample. Full development of this assay may lead to a test for this disease agent which would benefit the animal industries as well as those industries that produce pharmaceuticals and cosmetics from animal products.
Technical Abstract: Prion diseases or transmissible spongiform encephalopathies belong to a group of neurodegenerative diseases that infect both animals and humans. These diseases are associated with an accumulation of fibrils in the brains of infected individuals. These fibrils are composed of an abnormal isoform of a host-encoded glycoprotein that is characterized by its insolubility and partial resistance to proteases. Another characteristic of PrPsc is the wide range of pIs that have been observed on conventional isoelectrofocusing gels. In this study, we explored the use of capillary isoelectric focusing to characterize the isoelectric points for PrPsc isolated from sheep and hamster brain. We used a Beckman 5500 P/ACE using UV detection at 280nm. A cIEF 3-10 Kit from Beckman Instruments was used to perform the analysis. The PrPsc was solubilized in 0.01M Tris HCl, pH 8.00 containing 2mM EDTA, 5% SDS and 10% hexafluoroisopropanol at 100þC for r10 min. The solubilized PrPsc was placed over a high performance hydrophilic interaction column. After elution, the peaks were concentrated and assayed for immunoreactivity with specific antisera. The peaks that contained immunoreactivity were then placed on the cIEF capillary. The samples containing PrPsc were solubilized in 1% N-octylglucoside before isoelectric focusing. The pIs of the scrapie infected sheep sample had peaks representing pIs from 5.2 to 3.00 with a major peak at 3.09. The normal sheep brain had pIs that were higher. The hamster adapted scrapie strain had peaks representing pIs 6.47 to 3.8. These pIs were slightly higher than those obtained for the sheep samples. The use of cIEF to determine the pIs of PrPsc led to the new finding of the major species of prion protein from sheep with a very acidic pI.