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United States Department of Agriculture

Agricultural Research Service

Research Project: TRANSMISSION, DIFFERENTIATION, AND PATHOBIOLOGY OF TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES Title: Detection of PrPSc in Formalin Fixed Paraffin Embedded Tissue by Western Blot Differentiates Classical Scrapie, Nor98 Scrapie, and BSE

Authors
item Loiacono, Christie -
item Beckwith, Nadine -
item Kunkle, Robert
item Orcutt, Dennis
item Hall, S -

Submitted to: Journal of Veterinary Diagnostic Investigation
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 15, 2010
Publication Date: September 1, 2010
Repository URL: http://jvdi.org/cgi/content/full/22/5/684
Citation: Loiacono, C.M., Beckwith, N., Kunkle, R.A., Orcutt, D., Hall, S.M. 2010. Detection of PrPSc in formalin-fixed, paraffin embedded tissue by Western blot differentiates classical scrapie, Nor98 scrapie, and bovine spongiform encephalopathy. Journal of Veterinary Diagnostic Investigation. 22(5):684-689.

Interpretive Summary: Transmissible spongiform encephalopathies (TSEs) including bovine spongiform encephalopathy (BSE) and sheep scrapie are fatal brain disorders associated with the presence of infectious proteins called prions (PrP**Sc). Identifying PrP**Sc in the brain is typically based upon tests such as immunohistochemistry (IHC) which uses formalin fixed paraffin embedded tissue (FFPET) or western blot (WB) which uses fresh/frozen, non-formalin fixed tissue. Either assay can discriminate between BSE, classical scrapie and a previously reported strain of scrapie recently identified in the US named Nor98 scrapie. Separate tissue samples are required from the same animal to run these two different assays. This may result in inconsistent test results for the same animal. Sampling problems such as collecting insufficient volumes of fresh tissue or less than the optimal piece of brainstem for IHC can affect the ability of the test procedures to offer definitive and discriminatory results. Recently, a WB method using FFPET has been developed which successfully identified PrP**Sc in sheep affected by classical scrapie. Here we describe the use of this technique to produce discriminatory WB results identifying classical BSE and both classical and Nor98 scrapie using paraffin embedded brain samples. FFPET WB protein banding patterns were similar to protein banding patterns from WB assays utilizing fresh tissues from the same animals and results correlated well with the IHC test results from these animals. Since tissues embedded in paraffin remain remarkably stable in long-term storage, and it is commonplace for pathology laboratories to retain vast libraries of FFPET as reference materials, the findings presented here offer an expanded opportunity to conduct retrospective studies on PrP**Sc strains and TSE origins.

Technical Abstract: Transmissible spongiform encephalopathies including bovine spongiform encephalopathy and scrapie are fatal neurodegenerative disorders associated with the presence of an infectious abnormal isoform of normal mammalian proteins called prions (PrP**Sc). Identification of PrP**Sc in the CNS is typically based upon immunoassays including immunohistochemistry (IHC) using formalin fixed tissues or western blot assays (WB) using fresh/frozen, non-formalin fixed tissues. Each assay can discriminate between BSE, classical scrapie and a previously reported strain of scrapie recently identified in the US named Nor98 scrapie. Separate tissue samples are required from the same animal to run these two different immunoassays. This may result in inconsistent test results for the same animal. Sampling problems such as collecting insufficient volumes of fresh tissue or less than optimal anatomic location of brainstem for IHC can affect the ability of the test procedures to offer definitive and discriminatory results. Recently, a WB method using formalin-fixed paraffin embedded tissue (FFPET) to identify PrP**Sc has been developed which successfully identified PrP**Sc in sheep affected by classical scrapie. Here we describe the use of this technique to produce discriminatory results identifying classical BSE and both classical and Nor98 scrapie using paraffin embedded brain samples. FFPET WB protein banding patterns were similar to protein banding patterns from WB assays utilizing fresh tissues from the same animals and results correlated well with the IHC PrP**Sc positive staining present in the cerebellum and obex regions of brain samples from these animals.

Last Modified: 10/22/2014
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