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ARS Home » Research » Publications at this Location » Publication #247752
Title: Generation of Monoclonal Antibodies to Purified Prions in Lipid Rafts

Author
item Hnasko, Robert
item Stanker, Larry
item Lin, Alice

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 10/20/2009
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: Prion diseases are fatal neurodegenerative disorders caused by prion proteins (PrP). Infectious prions accumulate in brain through a template mediated conformational conversion of endogenous PrPC into alternately folded PrPSc. Immunoassays toward pre-clinical detection of PrPSc have been confounded by low-level accumulation in non-neuronal tissue and the lack of PrPSc selective antibodies. We define a method to purify infectious PrPSc from biological tissues for use as an immunogen and immunoassay antigen. Significant prion enrichment is accomplished by sucrose gradient centrifugation of infected tissue and isolation with detergent resistant membranes (DRMs) from lipid rafts. Purification of PrPSc from DRMs was accomplished using phosphotungstic acid protein precipitation after proteinase-K (PK) digestion followed by size exclusion chromatography to separate 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. Hybridomas producing anti-prion monoclonal antibodies were identified by a direct ELISA screen comparing supernatant binding to PK-treated PrPSc DRMs versus PrPC DRMs. We demonstrate the utility of purified PrPSc in lipid rafts as both an immunogen in Prnp0/0/Balb/cJ mice and antigen for selection of hybridomas producing high-affinity anti-prion monoclonal antibodies.