2008 Annual Report
1a.Objectives (from AD-416)
To develop new reagents and methods for the detection and evaluation of prion proteins in animal and environmental samples.
1b.Approach (from AD-416)
Bovine Spongiform Encephalopathy (BSE) and other transmissible prion diseases represent an important agricultural issue and pose unique diagnostic challenges. Unlike conventional microbes, prions do not require agent-specific nucleic acid in order to multiply. Propagation occurs in infected animals when normal prion protein (PrPc) becomes “misfolded” into an infectious form (PrPsc) in a template-driven process. No PrPsc specific probes have been developed and conventional methods rely on differences in the sensitivity of PrPc from PrPsc to chemical reagents for detection. BSE has had a devastating impact on foreign agricultural economies and despite a U.S. agricultural ban on ruminant protein imports from these countries, BSE remains a bio-security threat. New methods for the sensitive detection of PrPsc in animals, their by-products and the environment are essential for preventing the transmission of disease. We will develop strategies for the extraction and enrichment of PrPsc for use in the development of novel diagnostic reagents and platforms. These models will be used for the assessment of low-level PrPsc in pre-clinical animals and the environment. Methods will focus on isolation of PrPsc in detergent resistant membranes, utilization of sensitive transgenic mice as models of infectivity, cell based bioassay, and production of monoclonal antibodies. The generation and establishment of new reagents and detection platforms will provide the necessary diagnostic tools to achieve early detection of low-level PrPsc in contaminated biological and environmental samples. Documents SCA with UCSF. Formerly 5325-32000-007-02S (4/08).
This year we developed a method for enrichment and purification of prion from brain homogenate. This novel and innovative approach yields a highly purified scrapie preparation for use in monoclonal antibody production and studies of prion structure. We generated and purified several new monoclonal antibodies following inoculation of mice with detergent resistant membrane preparation from brain homogenate enriched in prion. We have identified three hybridoma clones that produce antibody to a single 25kDa proteinase-K resistant protein by Western blot. In addition, we have shown that this protein shares similar biochemical properties with the prion protein, consistent with a glycosylated isoform. We are now working to definitively identify the primary structure of this 25kDa protein following immunoaffinity purification and are prepared to inoculate PrP-ablated mice with our purified prion preparation.
ADODR monitors this project through occasional visits across the San Francisco Bay, telephone conversations, and email with the Cooperator. ADODR and Cooperator conduct annual group meetings with all personnel, and occasionally meet at national scientific meetings for additional coordination.