Improvement of neutralizing activity of anti-canine parvovirus antibodies through directed evolution.

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(1)Characterization of monoclonal antibodies (mAb) to canine parvovirus (CPV)Five mAbs (CPla - 5a) reacting to CPV has characterized through ELISA, agglutination activity of antigen-coated latex microspheres, Western blotting analysis, inhibitory activity to CPV's hemagglutination (HI), neutralizing activity against CPV (VN). The amino acid sequence of complementarity determining region (CDR) of each mAb was analyzed through cloning of cDNA of each V domain. The amino acid sequence of CDR was classified three types, and concomitantly, the biological activity of the mAbs correlated to the sequence.(2)Expression of a canine immunoglobulin G (IgG) with recombinant baculoviruses.Previously, we have cloned cDNA of canine IgG. In this study, we expressed IgG by baculovirus vector system. To express the canine IgG, two systems were prepared. The one was a recombinant baculovirus which has dual promoter and expresses both heavy and light chain at the same time. The another was co-infection wit … More h two recombinant baculoviruses which independently express light or heavy chain. The amount of secreted IgG in both system was the same, around 0.1 μg/mL. The recombinant IgG can be purified with protein A column, together with heavy chains. The IgG was reacted with anti-IgG2 antisera (Bethyl Lab. Inc.), so this molecule is IgG2 subclass.(3)Construction of caninized antibody.As homology of amino acid sequence of frame work region to canine IgG is highest among the mAbs, CP2a was selected as a donor of the CDR sequence. The caninized CP2a antibody was expressed by baculovirus system and characterized its binding property with donor CP2a by Western blotting. The caninized CP2a reacted with CPV proteins equally with CP2a, in the same peptide region (219-292) of VP2 proteins.(4)Expression of single chain V fragment (scFv)ScFv molecules was constructed with CP1a because of its highest HI and VN activity, and expressed in baculovirus system. The expression was confirmed with Western blotting using histidine hexamer tag, but the HI and VN activities were not observed. To adapt the directed evolution method to improve the property of the antibody, more basic data on structure-activity relationship of these antibodies were necessary to establish the platform. Less