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Title: Mapping the topographic epitopes of a model antigen
Author: Paus, Didrik
ISNI:       0000 0001 3479 1566
Awarding Body: Open University
Current Institution: Open University
Date of Award: 2003
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The thesis describes the development of a method for determining the antigenicity of different regions of the surface of a protein. The method involved the construction of a set of mutant antigens in which cysteine residues had been introduced at different points in the surface of the antigen. Each mutant protein was tethered through the surface cysteine residue and a bifunctional chemical cross-linker to solid phase, allowing the creation of an array of mutant antigens, each oriented so as to expose or to mask different topological regions of the antigen surface. Indeed, as expected, binding of a set of monoclonal antibodies proved sensitive to the orientation of the antigen. By use of this array of oriented antigen it became possible to compare the different regions of the antigen surface for binding to rabbit antiserum, and thereby to create a topological map of the antibody response. When the immunisations were undertaken with use of Freund's adjuvant, the antibody response to β-lactamase was mainly directed against a region centered on a flexible loop. Furthermore it comprised antibodies cross-reactive to both native and denatured protein. By contrast, in the absence of Freund's adjuvant, the antibody response was more evenly distributed, and with few cross reactive antibodies. This suggested that Freund's adjuvant denatures β-lactamase (as was confirmed experimentally), and that the appearance of dominant epitopes may follow the presentation of denatured protein to the immune system. These observations have implications for understanding the basis for dominance of B-cell epitopes, and also for design of vaccines.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: Antigenicity