title: Acetone precipitation of the scrapie agent results in successful recovery of PrP(Sc) but decreased infectivity.
abstract: Bioassay is considered the most sensitive method for evaluating prion inactivation procedures. Because prions are resistant to methods effective at inactivating conventional microorganisms, prion inactivation research has focused on relatively harsh alternatives, such as concentrated sodium hypochlorite or sodium hydroxide. Often, bioassay for residual infectivity in these studies requires dilution or biochemical alteration of the treated sample in order to maintain subject health and survival. Ideally, prions from treated samples could be sufficiently separated from the inactivating agent without alteration of the sample and with negligible loss of infectivity prior to inoculation into the bioassay host. The current study was designed to evaluate acetone precipitation of the disease-associated form of the prion protein (PrP(Sc)) from brain homogenate derived from mice with the RML (Rocky Mountain Laboratory) strain of scrapie. The ability to recover PrP(Sc) was evaluated by Western blot. Dilutions of acetone-precipitated RML-positive brain homogenate were compared to nonprecipitated RML homogenate, resulting in similar PrP(Sc) detection levels down to 0.025 mg equivalents of brain tissue. The impact of the method on infectivity was investigated by bioassay in intracranially inoculated tga20 mice. Additionally, contributions to infectivity from the pellet and supernatant fractions were investigated. Acetone precipitation resulted in a 1-log₁₀ reduction in infectivity. Infectivity could not be reconstituted by the acetone soluble fraction of the infectious sample or uninfected brain. This study demonstrates that PrP(Sc) can successfully be precipitated out of infected brain homogenate using acetone but that there is a reduction in infectivity attributable to the procedure that would need to be considered when evaluating bioassay results.
authors.affiliation: Virus and Prion Research Unit, National Animal Disease Center, USDA, Agricultural Research Service, Ames, Iowa, USA.
authors.forename: J D
authors.initials: JD
authors.lastname: Smith
authors.forename: J J
authors.initials: JJ
authors.lastname: Greenlee
authors.forename: G H
authors.initials: GH
authors.lastname: Foster
authors.forename: E M
authors.initials: EM
authors.lastname: Nicholson
mesh_terms: D000096:Acetone / Q000737:chemistry; D000818:Animals; D001681:Biological Assay / Q000662:veterinary; D001921:Brain / Q000378:metabolism*; D011232:Chemical Precipitation; D003943:Diagnostic Techniques, Neurological / Q000662:veterinary*; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009474:Neurons / Q000737:chemistry*; D018620:PrPSc Proteins / Q000302:isolation & purification* / Q000472:pathogenicity*; D012608:Scrapie / Q000175:diagnosis* / Q000378:metabolism; D012997:Solvents / Q000737:chemistry
chemical_list: D018620:PrPSc Proteins; D012997:Solvents; D000096:Acetone