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ARS Home » Research » Publications at this Location » Publication #64791
Title: PARAMETERS AFFECTING A COMPETITION ASSAY FOR THE SCRAPIE PRION PROTEIN BY CAPILLARY ELECTROPHORESIS

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

Submitted to: Application High Performance Liquid Chromatography and Capillary Electropho
Publication Type: Abstract Only
Publication Acceptance Date: 11/25/1996
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: Transmissible spongiform encephalopathies of animals and humans are infectious diseases that cause degenerative disorders of the central nervous system. In these diseases, there is 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. In this study, we used synthesized peptides which defined antigenic sites on the prion protein and antibodies that were made to these peptides. We measured immunocomplex formation of these peptides and the antibodies by capillary electrophoresis using laser induced fluorescence for detection (CE-LIF). Two rabbit antisera were purified using protein G chromatography and affinity chromatography with the corresponding peptides. Fab fragments were made of the affinity purified antibodies. The two peptides spanning amino acids 89-103 and 142-154 were synthesized with an attached fluorescein at the N-terminal amino acid. A 20um I.D.x 20cm unmodified capillary and a 200 mM Tricine, pH 8.0 as the buffer were used. For CE-LIF, a voltage of 30kV and a current of 38 ma were used. Because of the increased efficiency in labeling, the sensitivity of the detection was increased by at least 10 fold over prior assays. The purity of the antibody also affected immunocomplex formation which was enhanced by the purification of the antibody and the production of the Fab fragments. One fmole of the unlabeled peptides and pg quantities of the prion protein from brain of scrapie infected sheep were detected.