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Purified preparations of scrapie prions contain a sialoglycoprotein of Mr 27,000-30,000, designated PrP 27-30, which is derived from the scrapie prion protein [Mr,33,000 Source of Scrapie Prions and Bioassay. A hamster-adapted isolate of the scrapie agent was passaged and prepared as described (1, 13).Preparation of the Subcellular Fractions. Weanling hamsters (LVG/LAK) were inoculated intracerebrally with 107 ID50 units of the scrapie agent. The brains were collected from hamsters sacrificed 60 days after infection and from age-matched uninfected animals. The brains were suspended in 0.32 M sucrose (10%, wt/vol) and homogenized with six bursts of 10 sec each by using a Polytron homogenizer set at medium speed. The homogenates were centrifuged in a Beckman 50.2 Ti rotor. Pellets were resuspended in 0.32 M sucrose solution and the volumes were adjusted to that of the supernatant. All solutions were kept on ice and all centrifugations were performed at 4°C. The fractions were adjusted to 10 mg of protein per ml with 0.32 M sucrose solutions. One-milliliter samples were centrifuged in the Beckman 50 Ti rotor at 38,000 rpm for 1 hr at 4°C. Pellets were resuspended and treated as described in Results. For digestion experiments, N-lauroylsarcosine (sarkosyl) was added from a 10% stock solution in 25 mM Tris'HCl/0.1 M NaCl, pH 7.4, to some of the samples. Control samples were diluted by the same amount of Tris buffer. Digestions were initiated by addition of an aliquot of proteinase K (2 mg/ml) in Tris buffer to give a final concentration of 0.1 mg/ml. After 30 min at room temperature, the digestions were terminated by addition of an aliquot of 0

Scrapie is a slow infection of the nervous system which progresses in the absence of any apparent immune response. The recent development of a large-scale purification protocol for scrapie prions made it possible to obtain substantial quantities of electrophoretically purified prion protein (PrP 27-30) and we report here on the successful production of a rabbit antiserum to PrP 27-30. The antiserum reacted with PrP 27-30 and several lower molecular weight proteins as shown by Western blots; it did not react with protein preparations from uninfected brains. Discrete structures in the subependymal region of scrapie-infected hamster brains were stained immunocytochemically. These same structures also stained with Congo red dye and showed green birefringence with polarized light, a characteristic of purified prion rods. This staining pattern suggests that they are amyloid plaques.

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Prion proteins (PrP) were localized in the brains of normal and scrapie-infected hamsters by immunohistochemistry and Western blotting. PrP monoclonal antibodies and monospecific anti-PrP peptide sera, which react with both the cellular (PrPC) and scrapie (PrPSc) isoforms of the prion protein, were used to locate PrP in tissue sections. In normal hamsters, PrPC was located primarily in nerve cell bodies throughout the CNS; whereas, in the terminal stages of scrapie, PrP immunoreactivity was shifted to the neuropil and was absent from most nerve cell bodies. Prion proteins were not uniformly dispersed throughout the gray matter of scrapie-infected hamster brains; rather, they were concentrated in those regions that exhibited spongiform degeneration and reactive astrogliosis. Since earlier studies showed that the level of PrPC remains constant during scrapie infection as measured in whole brain homogenates and no antibodies are presently available that can distinguish PrPC from PrPSc, we analyzed individual brain regions by Western blotting. Analysis of proteinase K-digested homogenates of dissected brain regions showed that most of the regional changes in PrP immunoreactivity that are seen during scrapie infection are due to the accumulation of PrPSc. These observations indicate that the tissue pathology of scrapie can be directly correlated with the accumulation of PrPSc in the neuropil, and they suggest that the synthesis and distribution of the prion protein has a central role in the pathogenesis of this disorder.