title: Generation of antisera to purified prions in lipid rafts.
abstract: Prion diseases are fatal neurodegenerative disorders caused by prion proteins (PrP). Infectious prions accumulate in the brain through a template-mediated conformational conversion of endogenous PrP(C) into alternately folded PrP(Sc). Immunoassays toward pre-clinical detection of infectious PrP(Sc) have been confounded by low-level prion accumulation in non-neuronal tissue and the lack of PrP(Sc) selective antibodies. We report a method to purify infectious PrP(Sc) from biological tissues for use as an immunogen and sample enrichment for increased immunoassay sensitivity. Significant prion enrichment is accomplished by sucrose gradient centrifugation of infected tissue and isolation with detergent resistant membranes from lipid rafts (DRMs). At equivalent protein concentration a 50-fold increase in detectable PrP(Sc) was observed in DRM fractions relative to crude brain by direct ELISA. Sequential purification steps result in increased specific infectivity (DRM <20-fold and purified DRM immunogen <40-fold) relative to 1% crude brain homogenate. Purification of PrP(Sc) from DRM was accomplished using phosphotungstic acid protein precipitation after proteinase-K (PK) digestion followed by size exclusion chromatography to separate PK and residual protein fragments from larger prion aggregates. Immunization with purified PrP(Sc) antigen was performed using wild-type (wt) and Prnp(0/0) mice, both on Balb/cJ background. A robust immune response against PrP(Sc) was observed in all inoculated Prnp(0/0) mice resulting in antisera containing high-titer antibodies against prion protein. Antisera from these mice recognized both PrP(C) and PrP(Sc), while binding to other brain-derived protein was not observed. In contrast, the PrP(Sc) inoculum was non-immunogenic in wt mice and antisera showed no reactivity with PrP or any other protein.
authors.affiliation: USDA Agricultural Research Service, Foodborne Contaminants Research Unit, Albany, CA, USA.
authors.forename: Robert
authors.initials: R
authors.lastname: Hnasko
authors.forename: Ana V
authors.initials: AV
authors.lastname: Serban
authors.forename: George
authors.initials: G
authors.lastname: Carlson
authors.forename: Stanley B
authors.initials: SB
authors.lastname: Prusiner
authors.forename: Larry H
authors.initials: LH
authors.lastname: Stanker
mesh_terms: D000818:Animals; D017951:Antigen Presentation; D015153:Blotting, Western / Q000379:methods; D001923:Brain Chemistry; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D006224:Cricetinae; D005260:Female; D007106:Immune Sera / Q000737:chemistry* / Q000276:immunology; D021962:Membrane Microdomains / Q000737:chemistry* / Q000378:metabolism; D008647:Mesocricetus; D051379:Mice; D008807:Mice, Inbred BALB C; D018345:Mice, Knockout; D010772:Phosphotungstic Acid; D018621:PrPC Proteins / Q000378:metabolism; D018620:PrPSc Proteins / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D000072002:Prion Proteins; D011328:Prions / Q000235:genetics
chemical_list: D007106:Immune Sera; D018621:PrPC Proteins; D018620:PrPSc Proteins; D000072002:Prion Proteins; D011328:Prions; C512657:Prnp protein, mouse; D010772:Phosphotungstic Acid