Detection of Prion Protein in Formalin-Fixed Brain by Hydrated Autoclaving Immunohistochemistry for the Diagnosis of Scrapie in Sheep

Abstract

4The diagnosis of scrapie has traditionally relied on histopatholog ic demonstration of characteristi c lesions in brain sections. In recent years, however, increasing attention has been given to the use of immunodiagnos tic methods that focus on detection of a specific protein known as the prion protein (PrP). 7 This protein, which is believed to play an essential role in the pathogenesis of scrapie, is present in normal brain as a protease-sensitive isoform (PrP-sen). In contrast, most of the PrP in scrapie brain is protease resistant (PrP-res), probably because of a posttranslational alteration affecting the conformation of normal PrP. An immunohistoch emical (IHC) method to detect PrP-res in periodate, lysine, paraformaldehyde (PLP)-fixed brains from sheep with scrapie has been described. 5 Researchers working on bovine spongiform encephalopathy have recently reported that PrP-res also can be detected in formalin-fixe d brain if tissue sections are autoclaved prior to immunostaining. 1 This novel method of antigen enhancement, termed hydrated autoclaving, was originally devised to improve the immunoreactivity of certain brain proteins. 10 Hydrated autoclaving has not been used to detect other antigens. However, antigen enhancement is believed to result from heat denaturation of the protein, and the same mechanism has been suggested as the basis for antigen retrieval methods that utilize microwave immunohistochemistry. 6 In the present report, we describe diagnosis of scrapie using hydrated autoclaving IHC on formalin-fixe d sheep brains. For comparative purposes, some brains also were tested for PrP-res by western blot assay or by IHC on PLP-fixed brain. Brain samples from 186 scrapie suspect sheep were submitted to veterinary medical colleges, state veterinary diagnostic laboratories, or the National Veterinary Services Laboratories. Additional brain samples from 10 sheep in a scrapiefree flock were used as controls. Tissue sections were cut from paraffin blocks of formalin-fixe d brain stem (usually obex or midmedulla) and mounted on positively charged