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
The titer of the scrapie agent was determined by measurements of time intervals from inoculation to onset of illness and from inoculation to death. Both intervals were found to be inversely proportional to the size of the dose injected intracerebrally into random-bred weanling Syrian hamsters. The logarithms of the time intervals minus a time factor were linear functions of the logarithm of the inoculum size. The time factors were determined by regression analysis in order to maximize these linear relationships. An equation relating the titer of the inoculum to the dilution of the sample and the length of the time intervals was developed. This equation facilitates the use of a computerized data base. Validation of these relationships was provided by comparing samples for which the agent was measured both by end-point titration and by time interval assay. Agreement between the two methods was generally within +/-0.5 log10 median infective dose units. No differences between the molecular properties of the agents from hamster and murine sources were observed using primarily the incubation time interval method with the former and end-point titration with the latter. The advantages of this new approach based on time interval measurements are considerable with respect to time and resources.
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