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Title: Cloning and bacterial expression of humanised anti-varicella-zoster virus antibody fragments
Author: Drew, P. D.
Awarding Body: University of Aberdeen
Current Institution: University of Aberdeen
Date of Award: 2000
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The aim of this research was to produce neutralising antibody fragments with potential for topical therapy of varicella-zoster virus (VZV) infections, due to their small size and expected improved penetration of dermal tissues. Murine medium cells were stably transfected with a previously constructed glutamine synthetase (GS) selectable plasmid encoding humanised anti-VZV glycoprotein H (gH) monoclonal antibody 206 (hu206 mAb). Several glutamine-independent clones were analysed for antibody production. One high-producing clone was selected for expansion and used for expression and antibody purification. This antibody was shown to be functional in enzyme-linked immunosorbent assay (ELISA), immunofluorescence (IF) and neutralisation assay (50% neutralisation concentration ~2 nmol l-1). The variable regions of hu206 mAb were cloned and expressed as single-chain antibody fragments (scAb) in Escherichia coli. Such fragments were shown to be functional in antigen binding ELISA and IF, but were not neutralising in vitro at concentrations tested (up to 2000 nmol l-1). scAb were made neutralising by pre-incubation with a secondary antibody, which would be expected to cause multimerisation. scAb with shortened linkers (5 residues or 0 residues), which would be expected to form multimeric diabodies or triabodies, had 50% neutralising concentrations of ~90 and ~700 nmol l-1 respectively. Such fragments were, however, less functional than the standard scAb in the binding ELISA. Fab fragments produced by papain digestion of hu206 mAb had similar binding profiles to the parental mAb in the binding ELISA, but were poorly functional in the in vitro neutralising assay (50% neutralising concentration >500 nmol l-1). Again, neutralising activity could be partially restored by pre-incubation with secondary antibody. Thus, these results suggest that multimerisation is important in neutralisation of VZV by antibody.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available