title: Specific binding of the pathogenic prion isoform: development and characterization of a humanized single-chain variable antibody fragment.
abstract: Murine monoclonal antibody V5B2 which specifically recognizes the pathogenic form of the prion protein represents a potentially valuable tool in diagnostics or therapy of prion diseases. As murine antibodies elicit immune response in human, only modified forms can be used for therapeutic applications. We humanized a single-chain V5B2 antibody using variable domain resurfacing approach guided by computer modelling. Design based on sequence alignments and computer modelling resulted in a humanized version bearing 13 mutations compared to initial murine scFv. The humanized scFv was expressed in a dedicated bacterial system and purified by metal-affinity chromatography. Unaltered binding affinity to the original antigen was demonstrated by ELISA and maintained binding specificity was proved by Western blotting and immunohistochemistry. Since monoclonal antibodies against prion protein can antagonize prion propagation, humanized scFv specific for the pathogenic form of the prion protein might become a potential therapeutic reagent.
authors.affiliation: Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana, Slovenia.
authors.forename: Nives
authors.initials: N
authors.lastname: Skrlj
authors.forename: Tanja
authors.initials: T
authors.lastname: Vranac
authors.forename: Mara
authors.initials: M
authors.lastname: Popović
authors.forename: Vladka
authors.initials: V
authors.lastname: Curin Šerbec
authors.forename: Marko
authors.initials: M
authors.lastname: Dolinar
mesh_terms: D000818:Animals; D000915:Antibody Affinity / Q000276:immunology; D000918:Antibody Specificity / Q000235:genetics / Q000276:immunology; D003198:Computer Simulation; D016503:Drug Delivery Systems / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D009154:Mutation; D011328:Prions / Q000276:immunology*; D015202:Protein Engineering / Q000379:methods*; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology*
chemical_list: D011328:Prions; D057127:Single-Chain Antibodies