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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

2015 Annual Report

1. Sample enrichment methods to improve prion detection by immunoassays. Infectious prions are highly enriched in lipid rafts and their isolation as detergent resistant membranes (DRM) from fresh or frozen samples results in soluble prion fractions suitable for both Western and enzyme-linked immunosorbent assays (ELISA)). The use of brain DRM fractions as compared to crude homogenates results in significant increase in the detection of infectious prions. In addition, the use of a strong chaotropic denaturant (guanidine-HCl) was shown to be effective in aldehyde fixed tissues to unmask anti-prion binding epitopes. Prion detection and localization in tissue sections has been improved by using these newly developed immunohistochemistry and histoblot methods.

2. Novel models and methods to generate improved anti-prion monoclonal antibodies. We have successfully generated five new anti-prion monoclonal antibodies by using chemically cross-linked prion protein as immunogen and genetically modified mice predisposed to the development of autoimmune diseases. The resulting anti-prion monoclonal antibodies exhibit unique biochemical properties that are suitable as in-solution capture antibodies necessary for the development of a highly sensitive sandwich enzyme-linked immunosorbent assay (ELISA). These antibodies bind prions from agriculturally relevant animals and are being used to develop rapid prion immunodiagnostic tests. This innovative prion antibody strategy has broad application to other target analytes that also exhibit limited immunogenicity.

3. A simple and fast immunoassay modification improves prion detection. We have developed a modified direct enzyme-linked immunosorbent assay (ELISA) for the simple and rapid detection of infectious prions from tissue samples. In this assay, a simple 10 minute treatment with the chaotropic denaturant guanidine-HCl effectively unmasked antibody binding epitopes resulting in increased antibody binding and assay sensitivity. This assay modification is compatible with antibody binding and reporter-mediated detection. Using this modified prion immunoassay, we have increased detection of proteinase-K resistant prions in animal models as early as 45 days post prion infection. This modified ELISA has been shown to enhance performance of a wide range of antibodies and facilitate the detection of other aggregate amyloid proteins.

Review Publications
Schmitz, M., Zafar, S., Silva, C.J., Zerr, I. 2014. Behavioral abnormalities in prion protein knockout mice and the potential relevance of PrPc for the cytoskeleton. Prion. 8(6) 381-386.
Hnasko, R.M. 2015. ELISA: Methods and Protocols. Methods in Molecular Biology. Volume 1318. New York: Humana Press. 435 p. doi: 10.1007/978-1-4939-2742-5.
Hnasko, R.M. 2015. The biochemical properties of antibodies and their fragments. In: Hnasko, R., editor. ELISA: Methods and protocols. Methods in Molecular Biology. 1st edition. New York, NY: Humana Press. p. 1-14. doi: 10.1007/978-1-4939-2742-5.
Hnasko, R.M., Stanker, L.H. 2015. Hybridoma technology. In: Hnasko, R., editor. ELISA: Methods and protocols. Methods in Molecular Biology. 1st edition. New York, NY: Humana Press. p. 15-28. doi: 10.1007/978-1-4939-2742-5.
Hnasko, R.M. 2015. Bioconjugation of antibodies to horseradish peroxidase (hrp).In: Hnasko, R., editor. ELISA: Methods and protocols. Methods in Molecular Biology. 1st edition. New York, NY: Humana Press. p. 43-50. doi: 10.1007/978-1-4939-2742-5.
Hnasko, R.M., McGarvey, J.A. 2015. Affinity purification of antibodies. In: Hnasko, R., editor. ELISA Methods and Protocols. New York, NY: Humana Press. p. 29-41. doi: 10.1007/978-1-4939-2742-5.
Hnasko, R.M., Hnasko, T.S. 2015. The western blot. In: Hnasko, Robert, editor. ELISA: Methods and protocols. Methods in Molecular Biology. 1st edition. New York, NY:Humana Press. p. 87-96. doi: 10.1007/978-1-4939-2742-5.
Stanker, L.H., Scotcher, M.C., Cheng, L.W., Ching, K., Mcgarvey, J.A., Hodge, D., Hnasko, R.M. 2013. A monoclonal antibody based capture ELISA for botulinum neurotoxin serotype B: toxin detection in food. Toxins. 5(11):2212-2226.
Mcgarvey, J.A., Hnasko, R.M., Stanker, L.H., Han, R., Connel, J. 2015. Bacterial populations on the surfaces of organic and conventionally grown almond drupes. Journal of Applied Microbiology. 119:529-538.
He, X., Patfield, S.A., Hnasko, R.M., Rasooly, R., Mandrell, R.E. 2013. A polyclonal antibody based immunoassay detects seven subtypes of Shiga toxin 2 produced by escherichia coli in human and environmental samples. PLoS One. 8(10):e76368.
Ching, K.H., He, X., Stanker, L.H., Lin, A.V., Mcgarvey, J.A., Hnasko, R.M. 2015. Detection of shiga toxins by lateral flow assay. Toxins. 7:1163-1173. doi: 10.3390/Toxins7041163.
Silva, C.J., Erickson-Beltran, M.L., Skinner, C.B., Dynin, I., Hui, C., Patfield, S.A., Carter, J.M., He, X. 2014. Safe and effective means of detecting and quantitating Shiga-like toxins in attomole amounts. Analytical Chemistry. 86(10):4698-4706.
Silva, C.J., Vázquez-Fernández, E., Onikso, B., Requena, J.R. 2015. Proteinase K and the structure of PrPse: the good, the bad, and the ugly. Virus Research. doi: 10.1016/j.virusres.2015.03.008.
Skinner, C.B., Patfield, S.A., Rasooly, R., Carter, J.M., He, X. 2013. Purification and characterization of Shiga toxin 2f, an immunologically unrelated subtype of Shiga toxin 2. PLoS One. 8(3):e59760.
Kong, Q., Wu, A., Qi, W., Qi, R., Carter, J.M., Rasooly, R., He, X. 2014. Electron-beam is effective in reducing Escherichia coli on blueberries and extending the shelf life of the fruits. Postharvest Biology and Technology. 95(2014):28-35.
Clotilde, L., Bernard Iv, C., Lin, A., Salvador, A., Lauzon, C., Muldoon, M., Xu, Y., Carter, J.M. 2015. Comparison of antibody- versus pcr-based assays for serotyping shiga toxin-producing escherichia coli recovered from various cattle operations. Foodborne Pathogens and Disease. 12(2):118-121.