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Research Project: Immunodiagnostics to Detect Prions and Other Important Animal Pathogens

Location: Produce Safety and Microbiology Research

2022 Annual Report

Transmissible spongiform encephalopathies (TSEs) are animal diseases caused by infectious prion proteins that result in a slow progressive neurodegeneration that is fatal. The observation that prion diseases can be transmitted between animals, including humans, by consumption of contaminated food necessitates strategies to mitigate their occurrence in the food supply. In support of existing public health and food safety measures the USDA conducts TSE surveillance of suspect animals to monitor the incidence of the disease in the livestock population. TSE diagnostic surveillance is dependent on the detection of infectious prions in post mortem brain samples by immunoassay. The limitations of current prion immunoassays necessitate the development of improved prion detection methods that can reliably monitor the: 1) effectiveness of the ruminant feed ban, 2) spontaneous occurrence of disease, and 3) zoonotic transmission of TSE from endemic reservoirs of disease. The objective of this research is to develop immunodiagnostic methodologies that improve the effectiveness of TSE surveillance of livestock. Objective 1: Define methodologies for tissue specific prion sample enrichment to increase immunoassay sensitivity. Subobjective 1.1: Define methods to enrich prions from fresh or frozen tissues. Subobjective 1.2: Define methods to enhance prion detection from aldehyde preserved tissues. Subobjective 1.3: Define methods to enrich prions from decomposed tissues. Objective 2: Generate and validate improved prion monoclonal antibodies to achieve increased selectivity and sensitivity. Subobjective 2.1: Inoculate Prnp(0/0) Balbc/J mice with purified infectious prions and perform hybridoma fusions to generate conformation specific anti-prion monoclonal antibodies. Subobjective 2.2: Characterize the biochemical properties and validate binding specificity of anti-prion monoclonal antibodies. Objective 3: Develop and deploy applied immunoassays for prion detection. Subobjective 3.1: Develop prion immunoassays and evaluate detection sensitivity in agriculturally relevant models. The project will generate transferable technologies useful in the diagnosis of TSEs and the detection of low-level infectious prions in livestock tissues. These technologies will facilitate ante mortem TSE detection tests that will enhance our understanding of TSE disease prevalence in captive and wild animal populations. An effective ante mortem test for prion disease would also be valuable in the diagnosis of the closely related human Creutzfeldt-Jakob disease (CJD) and aid in the discovery of effective therapeutic interventions.

To overcome the obstacle of detecting low-level prions as a result of slow prion propagation following initial infection and allow sampling of non-neuronal tissues for evaluation we will define methodologies for tissue specific prion sample enrichment. These enrichment methods will include the biochemical isolation of prions with lipid rafts from fresh or frozen tissue, the use of chemically mediated antigen retrieval from aldehyde fixed tissue, proteolytic degradation of interfering proteins from decomposing tissues, high molecular weight dialysis to retain large aggregate prion amyloid, and chemical precipitation to concentrate prion enriched samples. The application of these methods will result in an increased yield of prions from target tissues and improve the reliability of prion detection measures. The properties of prion antibodies dictate the sensitivity and selectivity of prion immunoassays used in the determination of disease status. To generate improved prion monoclonal antibodies (mAbs) we will use highly purified prion immunogens, genetically engineered prion-knockout mice, hybridoma technology, and stringent screening methods for the identification of high-affinity anti-prion mAbs. Identified mAbs will be evaluated for prion binding that includes: epitope mapping, affinity measurements, species and strain specificity, and immunoassay application. Rigorous selection criteria will be used to identify high-affinity conformation- dependent anti-prion mAbs for development of enhanced prion immunoassays. Effective and reliable TSE surveillance depends on the sensitive detection of infectious prions by immunoassay. Applied prion tissue enrichment methodologies along with improved anti-prion monoclonal antibodies will be used to develop and optimize immunoassays for prion detection. Construction and deployment of various immunoassay platforms and antibody conjugated reporters (enzymatic, colorimetric, and fluorescent) will address end user needs for sensitive tissue specific prion detection that include: enzyme-linked immunosorbant assay (ELISA), field deployable lateral flow immunoassay (LFIA), Western blotting (WB), and immunohistochemistry.

Progress Report
The project 2030-32000-009-000D entitled “Immunodiagnostics to Detect Prions and Other Important Animal Pathogens” was completed in 2018. That project was replaced by the bridging project 2030-32000-010-000D with no change in the project title, objectives or milestones. This bridging project is replaced by 2030-32000-0011-000D entitled “Rapid Antemortem Tests for the Early Detection of Transmissible Spongiform Encephalopathies and Other Animal Diseases” which began on March 16, 2022. For additional information, see the report for the new project. The primary objective of this project was to improve the detection of infectious prions from animal tissue by immunoassay. Despite difficulties spanning multiple years, the goals with three important innovations were accomplished. The first was the development of a series of anti-prion monoclonal antibodies that bind novel epitopes in the prion protein. These antibodies have proven to be effective tools for the sensitive detection of infectious prions in a variety of immunoassay formats. This work was in support of Objective 2. In support of Objective 1, the second innovation was the development of methodologies that improve prion recovery from tissue samples, thereby increasing the amount of available prion for detection. Also in support of Objective 1, the third innovation was the application of a chemical denaturant as an integrated step in a high-throughput microplate immunoassay that significantly enhanced antibody binding and prion detection. This also had the added benefit of inactivating prion infectivity, which minimized downstream biohazardous waste management. Together these three innovations were used to develop a post-mortem rapid enzyme-linked immunosorbent assay (ELISA) that can detect infectious prions in brain samples from asymptomatic animals as early as 24 days after infection. This work supported Objective 3. The impact of these efforts is underscored by issued U.S. patents and commercial license agreements that make our reagents available for diagnostic assay development. Furthermore, these reagents and methodologies were transferred to partners at the National Veterinary Services Laboratories responsible for transmissible spongiform encephalopathies diagnostic testing as part of ongoing prion disease surveillance.


Review Publications
Silva, C.J., Erickson-Beltran, M.L. 2022. Detecting differences in prion protein conformation by quantifying methionine oxidation. ACS Omega. 7(3):2649-2660.