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.
We have generated and characterized several novel anti-prion monoclonal antibodies that bind prions from multiple species. The use of these anti-prion monoclonal antibodies as part of immunodiagnostic assays has resulted in sensitive detection of prions from infected animals. We have filed a national and international patent application for this technology and have transferred these anti-prion immunoassays to the USDA-APHIS for use in their ongoing prion surveillance program. Our efforts have resulted in improved immunodiagnostic capability for the sensitive detection of infectious prions resulting in enhanced security of USA agricultural products. The ADODR and other ARS scientists communicate with scientists at the Cooperative's laboratory in San Francisco via frequent email exchanges, occasional telephone calls and trans-bay visits, and an annual private symposium hosted by the Cooperator.