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United States Department of Agriculture

Agricultural Research Service

Research Project: NOVEL STRATEGIES FOR THE DETECTION OF INFECTIOUS PRION PROTEINS
2010 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).


3.Progress Report

We have made significant progress in FY10 on this project having published two peer-reviewed manuscripts and submitted one patent application. To overcome the lack of immunogenicity of the infectious form PrPSc in wild-type animals we generated a transgenic mouse that lacks endogenous PrPC (normal prion protein) bred on a genetic Balbc/J background (Prnp0/0 Balbc/J). We demonstrated that wild-type mice fail to respond to a purified PrPSc immunogen whereas Prnp0/0 Balbc/J mice elicit a robust and specific immune response producing high-titer anti-prion antisera. To assist in the identification of hybridoma cells producing anti-PrPSc monoclonal antibodies we developed a screening method that can distinguish PrPSc from PrPC. Here we devised a differential comparative direct binding ELISA screen in high-throughput microtiter plates to favor identification of hybridomas producing conformational PrPSc selective monoclonal antibodies. We have used this screening methodology to successfully identify numerous hybridomas making high-affinity anti-prion monoclonal antibodies useful for PrPSc detection. Together we have generated and characterized eight new anti-prion monoclonal antibodies with differential binding properties to cellular prion proteins from different species. Five antibodies exhibit conformational dependent binding to discontinuous PrP epitopes and their binding is disrupted by reduction of the disulfide bond in the PrP protein. The high-affinity F4-31 anti-prion monoclonal antibody has increased the sensitivity of BSE prion detection (~500-fold) and will be useful in a wide range of immunoassays.

ADODR monitors this project through occasional visits, 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.


Last Modified: 10/24/2014
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