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

Location: Produce Safety and Microbiology Research

2018 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
This is the final report for this project that was replaced March 1, 2018 by the bridging project 2030-32000-010-00D, “Immunodiagnostics to Detect Prions and Other Important Animal Pathogens”. Please see the report for the new project for additional information. The overall objective of the project was to develop sensitive immunoassays for the detection of infectious prions to improve prion disease surveillance efforts. Novel reagents and methodologies that improve the detection of infectious prions from animal tissues have been published, patented and licensed. Significant accomplishments were made in hybridoma technology resulting in the generation of novel anti-prion monoclonal antibodies (Objective 2), tissue preparative methodologies that increase prion yields (Objective 1) and the development and application of a sensitive immunoassay for improved detection of infectious prions from asymptomatic animal tissue (Objective 3). Progress was made on all three objectives under NP 103. Under Objective 1, tissue preparation methodology was refined to further increase the content of infectious prion protein in a given sample. The association of the prion protein with lipid rafts in cellular membranes was exploited by sucrose density centrifugation, a methodology that allowed for substantive removal of interfering proteins while achieving a significant enrichment of infectious prions. The resulting lipid rich samples were found to be well suited for prion detection using conventional plate-based enzyme-linked immunoabsorbent assays (ELISA) or western blotting. Under Objective 2, binding epitopes of three anti-prion monoclonal antibodies were generated and characterized. The anti-prion monoclonal antibody (MAB) DRM2-118 binds near the N-terminus of globular prion with epitope binding occurring just after the proteinase-K cleavage site. This antibody binding site represents a unique epitope not previously described for other available anti-prion antibodies. Although the DRM2-118 MAB does not selectively bind the infectious prion protein isoforms it does exhibit preferential binding under certain assay conditions. This MAB has broad species specificity with demonstrated immunoassay applicability by ELISA, western blot, and in tissue section for the detection of infectious prions. In alignment with Objective 2 and 3, a novel approach has been defined and published to conclusively determine MAB variable regions (VR). Each VR protein sequence is unique, and the VR portion of an antibody is responsible for determining binding specificity to the target antigen. Identification of the MAB VR serves to define and preserve the identity of the antibody. Moreover, the proper expression of VR sequence can be used to generate novel nanobodies or single chain variable fragments (scFv) of small size with binding properties equivalent to the parent antibody. The exclusion of antibody regions that do not directly participate in antigen binding can minimize unwanted non-specific binding with complex sample substrates. These nanobodies are amenable to modification which affords reporter integration, increased stability and simplified production. These efforts serve to further enhance immunoassay detection sensitivities and offer added capabilities that expand assay platform applications. Under Objective 3, significant progress was made in the early detection of infectious prions by immunoassay. In these published experiments two important innovations are described that enhance prion detection in tissue from prion infected asymptomatic animals. A substantial enrichment of infectious prions is achieved when lipid rafts are collected from tissue samples. This methodology results in bulk removal of unwanted protein material and increased concentration of infectious prions. Comparisons of crude brain homogenates to lipid raft preparations clearly demonstrate the advantage of this enrichment step in improved prion detection sensitivity by ELISA and western blot. The second innovation is the integration of an assay compatible chemical denaturation step that disrupts the complex tertiary structure of prion aggregates. The consequence of this chemical denaturation step is an exponential increase in the available antibody binding epitopes that are normally masked by the tight aggregate prion amyloid status. The exposure of antibody binding sites by unraveling the infectious prions by denaturation results in an increase in antibody binding and improved prion detection by ELISA. An added benefit of this approach is a significant reduction in the infectivity of contaminated samples which facilitates biosafety and disposal of test materials. Importantly, normal endogenous prion protein resides as a monomer at steady state levels in uninfected animals as evaluated in mock time course experiments. As the normal prion protein is a monomer, and does not accumulate, its detection remains at a steady state basal level irrespective of chemical denaturation. However, the infectious prion protein exists as polymer aggregates of varied size composed of abnormal prion protein monomers packed and bound together. Following prion infection these abnormal prion polymers begin to accumulate both in total number and size as the disease progresses. The use of the chemical denaturant then functions to unravel these aggregates, exposing individual monomers, making more antibody binding sites available. So, the integration of a simple chemical denaturation step on either crude or enriched samples selectively increases the detection of the infectious prion protein. Consequently, the establishment of a basal steady state level of normal endogenous prion protein in tissue provides the basis for a threshold base assay to establish disease status. In our assay, the ability to unmask prion antibody binding epitopes with chemical denaturation reveals the presence of infectious prions when detection levels are above those established at the basal steady state for the endogenous prion from uninfected animals. This applies to both crude and enriched samples irrespective of proteinase-k pretreatment. Consequently, prion disease status can be effectively established from tissues harvested from asymptomatic animals.

1. New technology provides early detection of prion disease. Spontaneous occurrence of transmissible spongiform degeneration (TSE), a prion disease, can occur in agriculturally important ruminant populations. Although rare, the slow accumulation of infectious prions and associated disease symptoms necessitates strategies aimed at early detection to limit potential disease spread and contamination of the food supply. Conventional methods for prion detection rely on surveillance of targeted animal populations by identification of infectious prion protein in brain tissue. ARS researchers in Albany, California, developed an assay that detects the presence of infectious prion protein in the brain of animals after early infection and before the occurrence of disease symptoms. This methodology provides improved prion detection sensitivity thereby enhancing ongoing surveillance efforts to monitor disease prevalence.

2. Improved detection of atypical sheep scrapie. Selective sheep breeding programs have reduced the incidence of classical scrapie but revealed the existence of a new form of atypical scrapie. ARS scientists in Albany, California, developed a sensitive method for the detection of atypical scrapie from infected sheep. The results showed that atypical scrapie replication is independent of sheep breed. Consequently, breed specific scrapie eradication efforts fail to protect sheep from atypical scrapie infection. This methodology provides improved atypical scrapie detection thereby enhancing ongoing surveillance efforts to monitor disease.

3. Advancement in hybridoma technology toward better monoclonal antibodies. The monoclonal antibody (MAB) represents an essential tool for immunoassay detection used in diagnostics and as biological reagents. ARS scientists in Albany, California, have successfully developed the first bioassay for the determination and optimization of macrophage conditioned media (MCM) used as a culture supplement in support of monoclonal antibody producing hybridoma cells. A U.S. patent 9,868,940 was issued this year, which describes a novel cell line and bioassay for optimal MCM production as a cell culture supplement. This patent is supported by a manuscript published this year that demonstrates the impact of MCM use in hybridoma technology. These technologies are now available to antibody producers for the generation and selection of MAbs essential in the development of diagnostic immunoassays.

Review Publications
Hnasko, R.M., Lin, A.V., McGarvey, J.A., Stanker, L.H. 2018. Enhanced detection of infectious prions by direct ELISA from the brains of asymptomatic animals using DRM2-118 monoclonal antibody and Gdn-HCl. Journal of Immunological Methods. 456:38-43.
Sevillano, A.M., Fernández-Borges, N., Younas, N., Wang, F., Elezgarai, S.R., Bravo, S., Vázquez-Fernández, E., Rosa, I., Eraña, H., Gil, D., Veiga, S., Vidal, E., Erickson-Beltran, M.L., Guitián, E., Silva, C.J., Nonno, R., Ma, J., Castilla, J., Requena, J.R. 2018. Recombinant PrPSc shares structural features with brain-derived PrPSc suggesting that they have a similar architecture: Insights from limited proteolysis. PLoS Pathogens. 14(1):e1006797.
Silva, C.J., Erickson-Beltran, M.L., Martín-Burriel, I., Badiola, J., Requena, J.R., Bolea, R. 2017. Determining the relative susceptibility of four prion protein genotypes to atypical scrapie. Analytical Chemistry. 90(2):1255-1262.
Babrak, L.M., McGarvey, J.A., Stanker, L.H., Hnasko, R.M. 2017. Identification and verification of hybridoma-derived monoclonal antibody variable region sequences using recombinant DNA technology and mass spectrometry. Molecular Immunology. 90:287-294.
Silva, C.J. 2018. Food forensics: using mass spectrometry to detect foodborne protein contaminants as exemplified by Shiga toxin variants and prion strains. Journal of Agricultural and Food Chemistry. 66(32):8435-8450.
He, X., Ardissino, G., Patfield, S.A., Cheng, L.W., Silva, C.J., Brigotti, M. 2018. An improved method for the sensitive detection of Shiga toxin 2 in human serum. Toxins. 10(2):59.