Location: Produce Safety and Microbiology Research2013 Annual Report
1a. Objectives (from AD-416):
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.
1b. Approach (from AD-416):
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).
3. Progress Report:
This report documents progress for this new project which started April, 2013 and continues research from Project Number 5325-32000-008-00D “Detection of transmissible spongiform encephalopathy agents in livestock, wildlife, agricultural products and the environment”. Transmissible spongiform encephalopathies (TSEs) result in fatal neurodegenerative diseases caused by the exposure and accumulation of mis-folded prion proteins. Infected animals and their byproducts represent a threat to U.S. livestock populations, global economic trade and human health through the consumption of contaminated food products. The USDA maintains an ongoing TSE surveillance effort in targeted animal populations to establish the incidence of this disease in livestock, monitor rate changes, and ensure U.S. animal health. The current strategy of TSE diagnosis involves the evaluation of tissues from symptomatic animals using immunoassays for the detection of infectious prion proteins. These immunoassays depend on sensitive and selective antibodies against the prion protein that are capable of detecting and discriminating infectious prions in various tissue samples. ARS scientists in Albany, California, have generated and characterized a number of novel monoclonal antibodies that recognize prions in agricultural animal tissues. We have developed novel prion enrichment and retrieval methods to achieve enhanced immunoassay detection sensitivities that result in early detection of infectious prions from asymptomatic animals using multiple assay platforms. To address the need for a rapid TSE detection assay, ARS scientists have been working on the development of lateral flow immunochromatographic test strips to engineer a sensitive, easy to use, field deployable, sample tracking TSE lateral flow device.
1. Enhanced immunoassay detection of infectious prions using antigen retrieval. ARS scientists in Albany, California, have developed a modified direct binding enzyme-linked immunosorbent assay (ELISA) that integrates an assay compatible chaotropic agent as an antigen retrieval step that results in enhanced detection of infectious prions. This method can effectively increase the binding of anti-prion monoclonal antibodies resulting in improved assay signal-to-noise and a log order increase in sensitivity. Applying a similar technical innovation to aldehyde fixed tissue sections we were able to demonstrate improved anti-prion binding by immunofluroescent microscopy while maintaining intact tissue morphology. This immunofluorescent technique was transferred to our partners at the National Veterinary Services Laboratory (NVSL). The integration of this simple antigen retrieval step has broad impact on the performance of ELISA or immunohistochemical assays that use antibodies that bind large aggregate proteins associated with neurodegenerative disease.
2. Detection of infectious prions in asymptomatic animals. ARS scientists in Albany, California, have developed a modified prion ELISA that integrates both prion sample enrichment and retrieval of masked anti-prion antibody binding epitopes to achieve enhanced detection of infectious prions. This research integrates three technologies developed by ARS scientists in Albany, California, for improved detection of infectious prions. A high-throughput modified chemiluminescent ELISA was developed that uses: 1) prion enrichment of brain sample with lipid rafts, 2) chaotropic agent retrieval of prion antibody binding epitopes, and 3) anti-prion DRM monoclonal antibody directly labeled with a horseradish peroxidase reporter. Evaluation of brain samples from animals at different time points following intracerebral inoculation of infectious prions resulted in data that shows our modified ELISA is capable of detecting infectious prions in asymptomatic animals >20 days before the onset of disease symptoms with a significant improvement in signal-to-noise. These data are being prepared for publication and will impact the methodology used for prion detection by stakeholders.
3. Chemical modification of lysine to differentiate prion conformers. Infectious prion strains reflect abnormal tertiary protein structures that propagate and produce distinct disease pathology. ARS scientists in Albany, California, have developed a method of using small molecules to covalently bind lysine residues in protein samples and applied this technique to discriminate prion strains by immunoassay. Conformational differences in prion isoforms result in a distinct pattern of chemically available lysine residues for modification which differs from that of normal non-infectious prion protein. Anti-prion monoclonal antibodies with lysine residues as part of the binding epitope failed to bind normal prion proteins in chemically modified samples but retained binding to the abnormal prion isoforms. Antibodies were identified that can discriminate between specific prion strains by immunoassay. The impact of this approach on prion detection offers the potential to build immunoassays that allow for the direct detection of infectious prion protein without the need for an enzymatic removal of the normal isoform and identification of the prion strain.