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United States Department of Agriculture

Agricultural Research Service

Title: Detection of PrP**Sc in formalin-fixed tissues by western blot

Authors
item Nicholson, Eric
item Kunkle, Robert
item Hamir, Amirali

Submitted to: United States-Japan Cooperative Program in Natural Resources
Publication Type: Abstract Only
Publication Acceptance Date: November 1, 2006
Publication Date: November 1, 2006
Citation: Nicholson, E.M., Kunkle, R.A., Hamir, A.N. 2006. Detection of PrP**Sc in formalin-fixed tissues by western blot [abstract]. United States-Japan Cooperative Program in Natural Resources. p. 16.

Technical Abstract: Formalin fixation is the most prevalent form of tissue preservative. As such, formalin fixed tissue represents an important source of archival material for study. Formalin fixation requires little environmental control and preserves the cellular architecture of a wide range of tissues, an important aspect for the accurate and reliable diagnosis of transmissible spongiform encephalopathies (TSEs) by Immunohistochemistry (IHC), considered by some to be the gold standard for diagnosis of TSEs. Alternatively, freezing of tissue samples is ideal for Western blot analysis but results in disruption of cellular architecture. Both methods are employed in a comprehensive diagnosis of a TSE. Virtually all diagnostic methods for TSEs rely on immunodetection of the disease associated form of the prion protein (PrP**Sc). Since the prion protein is a host encoded protein, an animal affected with a prion disease will have both the normal cellular form of the prion protein (PrP**C) and PrP**Sc. To date no commercially available antibody can distinguish these two isoforms of the protein. IHC relies on a highly trained and experienced user to distinguish disease associated staining from background PrP**C while Western blot is dependent upon limited digestion of the sample with proteinase K removing PrP**C leaving only PrP**Sc. These two methods have different strengths and weaknesses and as such are best used in a complimentary manner. Under field conditions, the only available tissue samples may be formalin fixed. A method for detecting PrP**Sc in formalin fixed tissues would allow analysis of numerous archived samples by Western blot, would simplify preservation of field collected samples for TSE detection, allow both Western blot and IHC analysis of the same preserved sample, and allow adjacent regions of brain to be analyzed by both IHC and Western blot enhancing the study of TSEs Approaches to prepare formalin fixed tissues for Western blot suitable for various proteins have been reported, however, none are applicable to the detection of PrP**Sc as they employ conditions known to render PrP**Sc proteinase K sensitive. Here we present an approach that recovers the signal of PrP**Sc while retaining the associated proteinase K resistance such that PrP**C signal may be removed via proteinase K digestion.

Last Modified: 10/24/2014
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