Skip to main content
ARS Home » Pacific West Area » Albany, California » Western Regional Research Center » Produce Safety and Microbiology Research » Research » Research Project #424354

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

Location: Produce Safety and Microbiology Research

2014 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 improved 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 (IHC).

Progress Report
Transmissible spongiform encephalopathies (TSEs) are fatal neurological diseases caused by mis-folded prion proteins. TSE infected animals and their byproducts represent a threat to livestock populations, global economic trade and human health. Diagnosis of TSE disease, as part of USDA animal surveillance effort, is confounded by the limited accumulation of infectious prions in diagnostic tissues and the late onset of disease symptoms. For objectives 1 and 2, ARS scientists in Albany, California, have developed novel methods and reagents to: generate selective anti-prion monoclonal antibodies, increase the yield of infectious prions from target tissue samples and improve prion detection using sensitive immunoassay platforms. Progress has been made toward the generation of conformation dependent anti-prion monoclonal antibodies by the development and use of a recombinant luminescent prion-renilla protein reporter to facilitate anti-prion monoclonal antibody selection, the creation of a cell bioassay to define a macrophage culture supplement that improves monoclonal antibody producing hybridoma cell growth and survival and the generation of a novel fluorescent hybridoma cell line for use in the rapid isolation and growth of antibody producing cells. Using anti-prion monoclonal antibodies and modified enzyme-linked immunoassays developed by ARS scientists in Albany, California, we have shown improved detection of aggregate infectious prion proteins in the brain of infected asymptomatic animals. For objective 3, in collaboration with ARS scientists and industry partners, we continue our progress on the development and construction of improved immunoassay platforms that include rapid immuno-chromatographic and microfluidic assays for the detection of prions and other animal pathogens. The effort of ARS scientists to improve sample preparation, target antigen enrichment and immunoassays detection of infectious prions and other animal pathogens has resulted in partnerships with industry, speaking and writing invitations, patent applications, publications and technology transfer to numerous stakeholders.

1. Novel anti-prion monoclonal antibodies improve detection of infectious prions by immunoassay. Scientists from the Produce Safety and Microbiology Research Unit in Albany, California, have generated and characterized unique anti-prion monoclonal antibodies isolated from prion knockout mice using purified infectious prions. The prion binding properties of these antibodies have been extensively characterized by government, academic and industry partners and they have proven highly effective in the detection of infectious prions using multiple immunoassay formats used for disease surveillance. This technology has been transferred to, and validated by, a large international biotechnology partner through an MTA and a license for commercialization issued. These antibodies will be made available through this license to academic and industry partners for research and commercial purposes to improve our understanding of prion diseases and diagnostic detection strategies. The commercial distribution and marketing of these important anti-prion monoclonal antibodies to the broader research and industry communities achieves a major milestone by providing essential tools for the construction and deployment of effective immunoassays used in prion disease surveillance.

2. Methods to isolate and detect prions in decomposed brain samples. Isolating and detecting prions in decomposed tissue is often necessary to identify infected animals post mortem. Scientists from the Produce Safety and Microbiology Research Unit in Albany, California, have developed effective methods for isolating and detecting infectious prions from degraded brain samples useful for disease diagnosis. These methods provide an alternative to conventional immuno-histochemical methods that perform poorly on decomposed samples. The use of these methods will ensure proper prion disease diagnosis from dead animals where ongoing decomposition confounds prion surveillance efforts.

3. Synthesis of small molecule reagents to detect prions. ARS scientists from the Produce Safety and Microbiology Research Unit in Albany, California, have synthesized small molecule reagents that selectively modify infectious prion proteins. These reagents covalently bind to locations present only in abnormal infectious prions. This permits the selective immunodetection of infectious prions in the presence of the normal cellular PrP isoform. These reagents have been transferred to an ARS collaborator in Germany for further evaluation.

4. Identification of peptides that selectively bind infectious prions. ARS scientists in the Produce Safety and Microbiology Research Unit in Albany, California, have identified numerous peptides that can selectively bind either the normal or abnormal conformations of the prion protein. These peptides are derived from the functional domains of larger parent proteins that are involved in intracellular protein transport. The immobilization of these synthetic peptides on a microarray membrane can be used for the discrimination of prion isoforms from tissue samples when probed with anti-prion monoclonal antibodies labeled with a reporter. The construction of a peptide immunoarray can be used for the effective detection of infectious prions without the need of sample pretreatment to remove endogenous cellular PrP. In this invention we are the first to describe and demonstrate the direct detection of infectious prions from that of native PrP protein using specific prion interactions with peptides that discriminate prion isoforms. This invention provides a simplified and rapid test to identify the presence of infectious prions in environmental samples and target animal tissues resulting improved prion disease surveillance. A patent has been issued for these novel peptide sequences - U.S. Patent No. 8,541,166.

Review Publications
Silva, C.J. 2014. Applying the tools of chemistry (mass spectrometry and covalent modification by small molecule reagents) to the detection of prions and the study of their structure. Prion. 8(1):42-50. DOI:
McGarvey, J.A., Connell, J., Stanker, L.H., Hnasko, R.M. 2014. Bacterial population structure and dynamics during the development of almond drupes. Journal of Applied Microbiology. 116:1542-1552.
Schmitz, M., Greis, C., Ottis, P., Silva, C.J., Schulz-Schaeffer, W., Wrede, A., Koppe, K., Onisko, B., Requena, J.R., Govindarajan, N., Korth, C., Fisher, A., Zerr, I. 2014. Loss of prion protein leads to age-dependent behavioral abnormalities and changes in cytoskeletal protein expression. Molecular Neurobiology. DOI: 10.1007/s12035-014-8655-3; 50(3):923-936.