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ARS Home » Research » Publications at this Location » Publication #60557
Title: 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 - USDA-APHIS-NVSL,AMES,IA

Submitted to: Journal of Microcolumn Chromatography
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/2/1995
Publication Date: N/A
Citation: N/A

Interpretive Summary: Scrapie is a disease in sheep that causes their central nervous system to degenerate and, eventually, the sheep die. Although this disease has been described over two hundred years ago, there is still no diagnosis available until the animal shows clinical signs that lead to death. There is evidence that a normal protein found on nerve cells is altered and this, in turn, begins the disease process. This altered protein is present in small amounts. The present tests can diagnose scrapie after death of the animal. In order to understand the disease process, it is necessary to have a test to diagnose scrapie before death. Recently, a new technique based on the mobility of proteins in an electrophoretic field has been developed. We used this technique, called capillary electrophoresis, to detect the presence of this altered protein in brain samples from scrapie infected sheep.

Technical Abstract: Transmissible spongiform encephalopathies of animals and humans are infectious diseases that cause progressive degenerative disorders of the central nervous system. These diseases are caused by a post translational modification which truncates the normal host cellular protein at the N-terminus of the protein. This modification makes the protein resistant to proteases and causes it to form rod-shaped fibrils in the brains of infected animals. The monomeric form (prion protein) of these aggregated fibrils has a molecular mass of ca.27kdaltons. Using this information we investigated the use of capillary electrophoresis as a diagnostic method. A synthesized peptide from the prion protein was labeled with fluorescein iodoacetamide. When the fluorescein labeled peptide was incubated with increasing concentrations of the rabbit antibody, a new peak that was proportional to the amount of antiserum in the reaction mixture with a concurrent reduction in the labeled peptide peaks was present on the electropherogram. Incubation overnight at 4degreesC enhanced immunocomplex formation. Competition studies were carried out using a concentration of rabbit antibody that was ca.50% of the maximum amount of immunocomplex formation. Addition of unlabeled peptide as well as material prepared from scrapie infected sheep brain but not material from normal sheep brain reduced the amount of immunocomplex formation observed on the electropherogram. We concluded that capillary electrophoresis can be used to detect the scrapie prion protein.