Detection of Scrapie-associated Fibril (SAF) Proteins Using Anti-SAF Antibody in Non-purified Tissue Preparations

Abstract

Antisera raised to scrapie-associated fibril (SAF) proteins were used to detect scrapie-specific polypeptides in three different non-purified brain preparations: a synaptosomal-mitochondrial fraction, 20% brain homogenate and 20% brain homogenate extracted with Sarkosyl. The concentration of SAF proteins in the preparations was greater than the quantity of SAF as detected by negative stain electron microscopy. This suggests that not all of the protein exists in the form of SAF. An immunologically reactive 33K to 35K protein was detected in both normal and scrapie brain preparations. This protein was susceptible to complete proteinase K (PK) digestion in normal brain preparations and it is suggested that scrapie infection is responsible for post-translational modifications which confer PK resistance in scrapie preparations. These modifications may also play a role in the antigenic differences seen in a variety of scrapie agents. SAF-specific proteins were also detected in the spinal cords and spleens from scrapie-affected animals. Detergent extraction of material followed by PK treatment and Western blot analysis is a highly specific and sensitive method for the detection of SAF proteins. This procedure could be applied to human neurological diseases of unknown aetiology.