|SERBAN, ANA - University Of California|
|CARLSON, GEORGE - McLaughlin Research Institute|
|PRUSINER, STANLEY - University Of California|
Submitted to: Prion
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/9/2010
Publication Date: 6/9/2010
Citation: Hnasko, R.M., Serban, A.V., Carlson, G.A., Prusiner, S.B., Stanker, L.H. 2010. Generation of Antisera to Purified Prions in Lipid Rafts. Prion. 4:294-104.
Interpretive Summary: Prions cause fatal brain diseases in humans and animals and can be transmitted by consumption of contaminated meat and byproducts. Current tests fail to detect prion infection in animals before they show signs of illness. Here we report a simple and effective method to enrich prions in biological tissue and fluids to increase detection sensitivity. We also present a method to generate antibodies useful for prion diagnosis. Such improved sample preparation and detection sensitivity will increase reliability of prion detection and reduce the risk of disease transmission.
Technical Abstract: Prion diseases are fatal neurodegenerative disorders caused by prion proteins (PrP). Infectious prions progressively accumulate in brain through a template mediated conformational conversion of endogenous host PrPC into alternately folded protein PrPSc. A diagnostic test with sufficient sensitivity to detect PrPSc in pre-clinically infected animals has proven difficult and there remains a need to develop PrPSc selective antibodies for use in a rapid and robust immunoassay. Antibody generation has been confounded by difficulty in isolation of pure prion antigen and its lack of immunogenicity in wild-type (wt) animals. Here we define a simple method to enrich and isolate infectious PrPSc from brain homogenate that is suitable for use as an immunogen. Prion enrichment is accomplished by sucrose gradient centrifugation of infected brain homogenate and isolation with detergent resistant membranes from lipid rafts (DRMs). Treatment with proteinase-K (PK) effectively digests PrPCC and other DRM proteins and size exclusion chromatography is then used to separate PK-resistant high molecular weight PrPSc aggregates from PK and protein fragments. Phosphotungstic acid (PTA) is then used to precipitate protein resulting in a concentrated highly purified prion preparation that retains infectivity. Immunization with purified PrPSc antigen was performed using 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 PrPS, 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 brain derived protein. Here we demonstrate an effective method for the enrichment and purification of infectious prions devoid of PrPC and other brain proteins. Moreover, we show differences in the immunogenicity of purified PrPSc following inoculation of wt versus Prnp0/0 mice. The use of purified PrPSc in lipid raftas as immunogen in Prnp0/0/Balb/cJ mice provides a new approach toward the generation of selective high-affinity antibodies against prion proteins.