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United States Department of Agriculture

Agricultural Research Service

Title: Evaluation of a Competitive Immunoassay for the Prion Protein Using High Performance Size Exclusion Chromatography

Authors
item Schmerr, Mary Jo
item Kohliekova, M - INST CHEM TECH, CZECH REP
item Alpert, A - POLYLC INC, COLUMBIA, MD

Submitted to: Separations in the Biosciences International Symposium
Publication Type: Abstract Only
Publication Acceptance Date: July 25, 2001
Publication Date: N/A

Technical Abstract: The abnormal prion protein is the putative agent of transmissible spongiform encephalopathies. A practical diagnostic test with the potential of high throughput is needed for this group of fatal, neurodegenerative diseases. A competitive assay has been developed using fluorescein-labeled peptides from the prion protein and corresponding antibodies. Capillary electrophoresis (CE) using laser induced fluorescence was the test platform for the analysis. Another test platform using a different method of analysis was desirable, because the CE method was the only available assay for detecting the prion in blood of naturally infected animals and humans. We chose to develop high performance size exclusion chromatography (HP-SEC) as another method to perform the analysis. Both methods (CE and HP-SEC) were able to separate the antibody-bound peptide from the free peptide, permitting them to be measured directly. Because the sampling volume required is very small for CE, the sample could be split and analyzed by HP-SEC. Standard competition curves were generated using the unlabeled peptide and recombinant bovine prion protein as competitors. Blood samples prepared from sheep with clinical scrapie and from normal sheep were analyzed. The results obtained by HP-SEC were similar to those obtained by CE. The lower sensitivity of the HP-SEC method compared to the CE method was overcome by increasing the volume of the sample. The HPLC-SEC method could detect the prion protein in the high picogram-range quantities from approximately 15 ml blood of naturally infected animals and humans. Further development of these two methods may lead to a diagnostic test for pre-clinical stages of these diseases.

Last Modified: 4/20/2014
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