Ijaz, Samreen; (1998) Production and use of monoclonal antibodies against a conserved epitope of hepatitis B surface antigen. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The hepatitis B surface antigen (HBsAg) displays the major neutralising epitope of the hepatitis B virus (HBV) termed the a determinant. As well as its use as an immunogen in vaccines, HBsAg is the primary diagnostic marker for HBV, with its detection in serum being used for routine screening in diagnostic laboratories. Recently, mutations within the a determinant have been described, some of which have been linked to false- negative HBsAg reactions in the serum of HBV infected individuals. The inability of some assays to detect HBsAg mutants will result in the failure to monitor immune responses as well as the transmission of infection through blood or organs as a result of inaccurate diagnosis. This thesis describes the use of monoclonal antibodies (MAbs) to define epitopes present on the wild-type (WT) HBsAg, that are preserved on HBsAg carrying mutations in the a determinant. The MAbs were raised against mutant HBsAg purified from two renal transplant patients. Following the screening process, those hybridomas which recognised the wild type and mutant HBsAg were selected. Characterisation studies using recombinant HBsAg further demonstrated that the MAbs were able to detect mutants ranging from codon 133 to 145. Once formatted into a solid phase radioimmunoassay, the MAbs were used to detect naturally-occurring in vivo mutant-virus infections. The identification of the conserved epitope in the first loop of the a determinant, using oligopeptides, highlighted possible limitations of the monoclonal antibody which were later demonstrated in a study assessing the prevalence of HBsAg mutants in orthotopic liver transplant patients. Changes associated with natural genetic variation in the first loop resulted in the monoclonal antibody failing to recognise HBsAg in specimens from patients infected with viruses of subtype ayw3 carrying changes in the putative binding site at residues 125 and 127. An important, although maybe not pan-reactive, epitope has been identified which can be used to improve HBsAg detection assays as well as providing a strategy for studying the prevalence of HBsAg mutants in different populations.

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