Detection of the Disease-Associated Isoform of the Prion Protein in Formalin-Fixed Tissues by Western Blot

Abstract

Abstract. Clinical signs of prion disease are not specific and include a variety of differential diagnoses. Serological tests and nucleic acid-based detection methods are not applicable to prion-disease-agent detection because of the unusual nature of the infectious agent. Prion-disease diagnosis is primarily conducted by means of immunodetection of the infectious agent, typically by at least 2 distinct procedures with immunohistochemistry and Western blot being the most informative. These approaches differ in the need for formalin-fixed and frozen or fresh tissue respectively. This work describes a method for the detection of the disease-associated isoform of the prion protein by Western blot using formalin-fixed tissues. The approach requires only minimal modification of existing Western-blot procedures and could readily be incorporated into existing detection schemes for confirmatory purposes when fresh or frozen tissues are unavailable.

Enrichment of PrPSc in formalin-fixed, paraffin-embedded tissues prior to analysis by Western blot

“…Studies of formalin-fixed tissues report a marked sensitivity decrease for tissues left in formalin for 2 or more years. 3 The method described herein is an extension of previously published methods for WB of formalin-fixed samples for the purposes of detecting the disease-associated form of the prion protein. 1,3 From each paraffin block, 4 tissue sections (each 5-µm thick) were collected into 1.5-ml microfuge tubes.…”

“…While typical WB procedures employ fresh or frozen tissues, recent advances allow WB analysis of PrP Sc from tissues that had been fixed in formalin expanding the opportunity to analyze TSE isolates for which no fresh or frozen tissues were available. 1,3 Using this approach, formalinfixed, paraffin-embedded tissues have been used in WB-based molecular profiling differentiating TSE strains. 2 The method presented in the current study enhances the sensitivity of these methods using centrifugation to pellet all detectable PrP Sc , allowing substantially more tissue to be analyzed by WB.…”

Paraffin-embedded tissue blot as a sensitive method for discrimination between classical scrapie and experimental bovine spongiform encephalopathy in sheep

“…Western blot usually requires fresh or frozen material, although protocols do exist that permit processing of fixed material. 12 This, together with the DPET blot, offer scope to extend discriminatory testing into cases where the available tissue is limited to fixed material or is suboptimal. The method described has not been applied to any preclinical cases to date.…”

Retrospective Analysis of Sheep Scrapie by Western Blotting with Formalin-Fixed Paraffin-Embedded (FFPE) Tissues

“…This is primarily because of geography, with Mongolia being a vast extent of land that includes both deserts to mountainous regions that can be subject to harsh climatic conditions. Nonetheless, pathological examinations are routinely used for the diagnosis of contagious diseases, including rabies and listeriosis.It has been reported that PrP Sc extracted from formalinfixed paraffin-embedded (FFPE) tissues can be detected by WB [12]. Because this technique could enable better TSE surveillance, we tested its usefulness on tissues from sheep scrapie cases for which frozen tissues were unavailable.…”

“…It has been reported that PrP Sc extracted from formalinfixed paraffin-embedded (FFPE) tissues can be detected by WB [12]. Because this technique could enable better TSE surveillance, we tested its usefulness on tissues from sheep scrapie cases for which frozen tissues were unavailable.…”