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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

2016 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
Management Unit reorganization along with resignation of personnel has resulted in critical vacancies to this program project from 2.6 scientific staff years at the onset of the project to the current 1.9 scientific staff years as well as a vacant full time technical support position. Despite these deficiencies, significant progress has been achieved in the program project objectives and milestones administered under National Program 103: Animal Health, Component 7, Transmissible Spongiform Encephalopathies. Using ARS developed anti-prion monoclonal antibodies and immunoassays we have demonstrated detection of infectious prions in animals prior to the onset of clinical symptoms using time-course samples. To achieve these results we developed a simple prion retrieval method compatible with rapid enzyme-linked immunoassays and immunohistochemical methods that results in enhanced detection of infectious prions. Prion infection results in the progressive accumulation of proteolytic resistant prion protein that is densely packed into aggregate amyloid fibrils. Despite the progressive accumulation of the abnormal prion, the conformation of these insoluble amyloids impedes detection methods. To overcome this obstacle, we developed an immunoassay method compatible with a strong chaotropic chemical denaturant that rapidly disrupts the abnormal prion amyloids and exposes previously blocked antibody binding epitopes resulting in enhanced detection. Importantly, in uninfected animals the normal native prion protein does not accumulate and the protein level is maintained at near constant rate. In these animals, detection of prion protein remains constant irrespective of the use of prion retrieval methods. However, in prion infected animals there are two pools of prion protein, the normal native prion and the abnormal prion that accumulates in densely packed amyloid. Here prion retrieval methods are necessary to detect the abnormal prion accumulated in the amyloid or detection is limited to the native prion protein and the infected animal appear to have prion protein levels similar to that of the uninfected. The importance of these observations is underscored by the fact that in uninfected animals’ prion protein levels remain constant and after prion infection, irrespective of strain, the total prion protein increases. Consequently the ability to detect the total prion protein from tissue samples allows the development of simple threshold based immunoassays to determine the disease status of the animal.

1. Chemically mediated prion retrieval results in enhanced prion detection by immunoassay. ARS scientists in Albany, California, have developed a high-throughput immunoassay method that results in the detection of infectious prions from brain tissue in asymptomatic animals. Application of a simple and assay compatible chaotropic agent on immobilized tissue extract results in disruption of aggregate prion amyloid structure, increased antibody binding and improved prion detection. In uninfected animals the total prion protein remains unchanged over time (steady-state), whereas in prion infected animals there is a progressive increase in the total prion protein that is accumulated as infectious abnormal aggregate prion protein amyloid. In conventional assays, the detection of infectious prion protein is hampered by its aggregate status, limited by available antibody binding epitopes, resulting in an under estimation of total prion protein present in a sample. In our modified direct enzyme-linked immunoassay (ELISA), the accumulated aggregate prion proteins are unmasked and increased detection, above normalized prion baseline level established from uninfected tissues, allows determination of animal disease status for the presence of a transmissible spongiform encephalopathy (TSE). Three of our anti-prion monoclonal antibodies used in this immunoassay have been patented, commercially licensed and are now available to the research community and industry stakeholders.

2. Covalent chemical modification of prion proteins alters anti-prion binding properties and facilitates the identification of infectious prion strains. Prion strains differ in abnormal conformation and exposed amino acid residues resulting in distinct enzymatic and antibody binding profiles. The use of small molecule reagents for covalent modification of exposed amino acid residues has facilitated the identification of prion strains associated with transmissible spongiform encephalopathies (TSEs). Unique structural differences in prion strains allows for specific modifications of anti-prion binding epitopes that allows for direct detection and discrimination of these strains by immunoassay. ARS scientists from Albany, California, in collaboration with German and Spanish scientists, have applied this approach in combination with immunoassay for the detection of distinct prion strains responsible for human Creutzfeld-Jakob Disease (CJD) and Fatal Familial Insomnia (FFI).


Review Publications
Silva, C.J., Erickson-Beltran, M.L., Skinner, C.B., Patfield, S.A., He, X. 2015. Mass spectrometry-based method of detecting and distinguishing type 1 and type 2 Shiga-like toxins in human serum. Toxins. 7:5236-5253.
Hnasko, R.M., Lin, A.V., Stanker, L.H., Bala, K., McGarvey, J.A. 2016. Prion extraction methods: comparison of bead beating, ultrasonic disruption and repeated freeze-thaw methodologies for the recovery of functional renilla-prion fusion protein from bacteria. In: Micic, M., editor. Sample Preparation Techniques for Soil, Plant, and Animal Samples. New York, NY: Humana Press. p. 389-399.
Silva, C.J., Erickson-Beltran, M.L., Dynin, I.C. 2016. Covalent surface modification of prions: a mass spectrometry-based means of detecting distinctive structural features of prion strains. Biochemistry. 55:894-902.
Llorens, F., Thune, K., Schmitz, M., Cramm, M., Tahir, W., Gotzmann, N., Zerr, I., Silva, C.J., Frau-Mendez, M.A., Ansoleaga, B., Berjawi, S., Carmona, M., Ferrar, I., Fernandez, I., Zarranz, J. 2016. Identification of new molecular alterations in Fatal Familial Insomnia. Human Molecular Genetics. Vol: 25; Page: 2417-2436.
Babrak, L.M., Lin, A.V., Stanker, L.H., McGarvey, J.A., Hnasko, R.M. 2016. Rapid microfluidic assay for the detection of botulinum neurotoxin in animal sera. Toxins. 8(1):13.