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Title: Conformation-Dependent High-Affinity Monoclonal Antibodies to Prion Proteins

item Stanker, Larry
item CLEVELAND, ELISA - University Of California
item Hnasko, Robert
item AZUCENA, LEMUS - University Of California
item SAFAR, JIRI - University Of California
item DEARMOND, STEPHEN - University Of California

Submitted to: Journal of Immunology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/12/2010
Publication Date: 6/7/2010
Citation: Stanker, L.H., Serban, A.V., Cleveland, E., Hnasko, R.M., Azucena, L., Safar, J., Dearmond, S.J., Prusiner, S.B. 2010. Isolation and characterization of new anti-PrP monoclonal antibodies. Journal of Immunology. 185: 729-737

Interpretive Summary: Bovine spongiform encephalopathy or BSE is a fatal neurological disease in cattle that was first described in the United Kingdom. The disease is caused by a prion or normal cellular protein that undergoes a change in shape resulting in the formation of large aggregates of the abnormal protein in the brain, cell death, and development of large vacuoles (spaces or holes) in the brain tissue. Conformation of the diagnosis of this disease currently is accomplished using immunoassays. Immunoassays rely on the ability of antibodies to specifically react with an antigen or pathogen, in this case binding to the prion protein in the brain. Current tests, while able to detect abnormal protein are not sensitive and rely on the digestion of normal protein with enzymes. This research describes development of a highly sensitive monoclonal antibody that substantially increases detection sensitivity. Incorporation of this antibody into prion diagnostics should provide producers and regulators with improved tools to identify and better manage BSE.

Technical Abstract: Prion diseases are fatal, neurodegenerative illnesses caused by the accumulation of PrPSc, an aberrantly folded isoform of the normal, cellular prion protein (PrPC). Detection of PrPSc commonly relies on immunochemical methods, a strategy hampered by the lack of antibodies specific for this disease-causing isoform. Here, we report the generation of 8 mAbs against PrP following immunization of Prnp-null mice with recombinant (rec) PrP. The 8 mAbs exhibited distinct differential binding to PrPC and PrPSc from different species as well as PrP-derived synthetic peptides. Five of the 8 mAbs exhibited binding discontinuous PrP epitopes, all of which were disrupted by addition of b-mercaptoethanol or dithiothreitol, which reduced the single disulfide bond found in PrP. One mAb F20-29 reacted only with human PrP while the F4-31 mAb bound bovine PrP; the KD values for both mAbs F4-31 and F20-29 were ~500 pM. Binding of all five conformation-dependent mAbs to PrP was inhibited by 2-mercaptoethanol in ELISA, Western blots and histoblots. One conformation-dependent mAb F4-31 was found to increase the sensitivity of an ELISA-based test by nearly 500-fold when it was used as the capture antibody. These new conformation-dependent mAbs were found be particularly useful in histoblotting studies, in which the low backgrounds after treatment with 2-mercaptoethanol created unusually high signal-to-noise ratios.