Generation of antisera to purified prions in lipid rafts |
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| Authors: | Robert Hnasko Ana V Serban George Carlson Stanley B Prusiner and Larry H Stanker |
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| Institution: | 1 USDA Agricultural Research Service; Foodborne Contaminants Research Unit; Albany, CA USA ;2 Institute for Neurodegenerative Diseases; University of California; San Francisco, CA USA ;4 Departments of Pathology and Neurology; University of California; San Francisco, CA USA ;3 McLaughlin Research Institute; Great Falls, MT, USA |
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| 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 PrPC into alternately folded PrPSc. Immunoassays toward pre-clinical detection of infectious PrPSc have been confounded by low-level prion accumulation in non-neuronal tissue and the lack of PrPSc selective antibodies. We report a method to purify infectious PrPSc 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 PrPSc 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 PrPSc 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 PrPSc antigen was performed using wild-type (wt) and Prnp0/0 mice, both on Balb/cJ background. A robust immune response against PrPSc was observed in all inoculated Prnp0/0 mice resulting in antisera containing high-titer antibodies against prion protein. Antisera from these mice recognized both PrPC and PrPSc, while binding to other brain-derived protein was not observed. In contrast, the PrPSc inoculum was non-immunogenic in wt mice and antisera showed no reactivity with PrP or any other protein.Key words: prion, scrapie, Prnp0/0 mice, purification methodology, antibody, antisera, lipid-rafts, detergent resistant membranes, neuroscience, immunization, diagnostic |
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