THE USE OF CAPILLARY SDS GEL ELECTROPHORESIS TO DETECT THE PRION PROTEIN EXTRACTED FROM SCRAPIE INFECTED SHEEP

Authors: Schmerr, Mary Jo; Cutlip, Randall; JENNY, ALLEN - USDA-APHIS-NVSL,AMES,IA

Submitted to: The Journal of Chromatography B: Biomedical Applications
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/17/1997
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 diseases 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 micrograms of brain sample. This is equivalent to about 10 billionths of an ounce. Full development of this assay may lead to a test for this disease agent which will benefit the animal industry as well as industries involved in using animal products to prepare pharmaceuticals and cosmetics.

Technical Abstract: Scrapie in sheep and in goats is the prototype of a group of transmissible spongiform encephalopathies (TSE). A feature of these diseases is the accumulation in the brain of rod shaped fibrils that form from an aggregated protein that is a protease-resistant form of a modified normal host cell protein. In this study, e compared SDS gel capillary electrophoresis to conventional SDS-PAGE to detect the monomer of this aggregated protein. This prion protein was extracted from the sheep brain by homogenizing the brain stem (10 percent w/v) in 0.32M sucrose and by using a series of ultracentrifugation steps and treatment with sodium lauroyl sarcosine and proteinase K. After the final centrifugation step, the pellet was resuspended in 10MM Tris pH 7.4 in a volume of equivalent of 0.1ml/g of brain used. This resuspended pellet was treated with 1 percent SDS and 5 percent 2-mercaptoethanol and boiled for 10 minutes. The analysis was done in a Beckman P/ACE 5500 using a SDS gel capillary (eCAP SDS 14-200 Beckman capillary). In infected sheep brain samples but not normal sheep, a major peak at a molecular mass of 16Kd and a minor peak with a leading shoulder were observed. Since the molecular mass determined for this protein was lower than that estimated on SDS-PAGE (22.4 Kdaltons), a Ferguson plot was made to determine if there were abberrations in the molecular mass determination. After correction, the major peak was estimated to be 19.5kD. This has a better correlation with that determined by SDS PAGE. The equivalent amount of brain sample in the capillary was approximately micrograms. For SDS-PAGE the amount of brain sample was 40- 50 mg. This is approximately 100 times less than that needed to do SDS- PAGE.