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United States Department of Agriculture

Agricultural Research Service

Title: Detection by Capillary Electrophoresis of the Binding of Fluorescent Peptides from Prion Protein to Prions Purified from Scrapie Infected Sheep Brain

Authors
item Schmerr, Mary Jo
item Jenny, Allen - APHIS,NVSL,PL,AMES,IOWA

Submitted to: Veterinary Virology International Congress
Publication Type: Abstract Only
Publication Acceptance Date: August 27, 1998
Publication Date: N/A

Technical Abstract: Transmissible spongiform encephalopathies of animals and humans are infectious diseases that cause degenerative disorders of the central nervous system. These diseases are caused by a post translational modification of a cellular protein found primarily on the surface of neurons. This modified protein (prion protein) aggregates into rod-shaped fibrils in the brains of infected animals and is resistant to proteases. The infected animals mount no observable immune response. Currently, there is a need for new methods to detect the presence of prion protein that requires less material than conventional methods. In this study, we used synthetic peptides which defined antigenic sites on the prion protein. These peptides which spanned amino acids 89-103 (pep1), 106-126 (pep2), 142-154 (pep3) and 167-178 (pep4) had fluorescein attached during synthesis at the N-terminal amino acid. We used laser induced fluorescence for detection (CE-LIF). A 20um I.D. x 20cm unmodified capillary and a 200 mM Tricine, pH 8.0 as the buffer were used. For CE-LIF, several voltages, incubation times and temperatures were used to determine the conditions for optimal binding. In this system, approximately 10 attomoles of the fluorescent labeled peptides could be detected. The prion protein was prepared from sheep brain using ultracentrifugation and high performance hydrophilic interaction chromatography. The fluorescent labeled peptides were then assayed for their ability to bind to purified prion protein. The binding of the fluorescent labeled peptides was observed by a shift in the fluorescence peak of the peptides. The labeled peptides showed significant shifts of the fluorescence peak when the prion protein was present. This shift was dependent on the concentration of the prion protein.

Last Modified: 4/17/2014
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