The aim of this project was to employ phage-display libraries to investigate epitopes recognised by antibodies directed against the hepatitis B virus surface antigen (HBsAg). The experiments were based upon a library comprising a pool of filamentous phage (fusion-phage) which displayed random hexapeptides on the surface of the particles. Antibodies of monoclonal and polyclonal origin were used for selection of phage that bound the antibodies via their exposed peptides. In the case of the monoclonal antibody, the phage ligands selected showed highly related amino acid sequences with a few distinctively conserved residues. These residues could be aligned with the primary sequence of the PreS1 domain of HBsAg thus precisely locating the epitope recognised by this monoclonal antibody. A quantitative assay, adapted specifically for use with fusion-phage particles, revealed binding constants in the nM range for the interaction of the fusion-phage with the antibody. Comparison of the binding constants of phage with divergent amino acid sequences and competition binding studies with synthetic peptides defined the epitope recognised by this antibody with high-resolution. A similar approach was applied to polyclonal serum IgG from a chimpanzee that had been vaccinated with HBsAg. It has been demonstrated that the phage-display approach can provide high-resolution epitope mapping for monoclonal antibodies. Quantitative values for the interaction in solution between fusion-phage representing this epitope and the antibodies have been obtained readily by means of a simple assay based upon phage titre. Further, even within the complex context of a polyclonal serum individual epitopes or at least elements of them could be identified, thus indicating immunologically important areas of this antigen.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:651397 |
| Date | January 1995 |
| Creators | Germaschewski, Volker |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/13893 |
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