specific binding of the pathogenic prion isoform development and characterization of a humanized single-chain variable antibody fragment致病朊病毒同种型发展和特征的特定绑定的变量人源化单链抗体片段.pdfVIP

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2017-09-12 发布于上海
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specific binding of the pathogenic prion isoform development and characterization of a humanized single-chain variable antibody fragment致病朊病毒同种型发展和特征的特定绑定的变量人源化单链抗体片段.pdf

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specific binding of the pathogenic prion isoform development and characterization of a humanized single-chain variable antibody fragment致病朊病毒同种型发展和特征的特定绑定的变量人源化单链抗体片段

Specific Binding of the Pathogenic Prion Isoform: Development and Characterization of a Humanized Single-Chain Variable Antibody Fragment ˇ 1 2 ´3 ˇ ˇ 1,2 1 Nives Skrlj , Tanja Vranac , Mara Popovic , Vladka Curin Serbec , Marko Dolinar * 1 Biochemistry Chair, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana, Slovenia, 2 Department for Production of Diagnostic Reagents and Research, Blood Transfusion Centre of Slovenia, Ljubljana, Slovenia, 3 Institute of Pathology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia 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. ˇ ˇ ˇ