Characterisation of anti-IgG monoclonal antibody A57H by epitope mapping.

Monoclonal antibodies have proved powerful tools in serological techniques and as probes for elucidating the antigenic determinants of macromolecules and cells. However an inherent problem of these reagents is the phenomenon of assay restriction, where a given antibody may perform well in some assays, whilst being poor or ineffective in others [I] . Thus stringent evaluation of a monoclonal antibody (MAb) in a number of assay systems is necessary. Furthermore, an appreciation of a target epitope is essential for developing a secure assay system e.g. capture enzyme linked immunosorbent assay (ELISA). Epitope mapping [2] permits identification of amino acid residues which may constitute a linear epitope recognised by a given MAb. By scanning sequential peptides of a protein of interest, the location of key residues involved in antibody-epitope interactions may be ascertained. In generating MAbs to allotypic determinants on the human IgGl subclass, female balbic mice were immunized with a purified pFc’ fragment of a human heavy chain disease protein PER: typed GI m(a,x). Subsequent fusion with NSO myeloma cells generated 71 hybridomas with anti-IgG reactivity. Culture supernatants (and latterly ascites) were screened in haemagglutination, (HA), haemagglutination inhibition and ELISA [I] . One hybridoma, A57H (isotype: IgM) showed pan-IgG teactivity when tcsted against a panel of IgGI-4 myeloma proteins in both assay systems. The epitope recognised by MAb A57H was particularly robust to physicochemical degradation (Table 1) and recognised a linear epitope since the antibody detected the pFc’ fragment in Western blotting 131. In epitope mapping investigations, biotinylated 15 mer peptides (offset of 2) spanning the lgGl CH2 and CH3 regions, were immobilized in solid-phase immunoassay by streptavidin. Binding of MAbs was visualised using HRPlabelled goat anti-mouse conjugate and ABTS as substrate. MAb A57H recognised a set of peptides in the CH3 region, Table 2. The effect of physicochemical degradation on MAb target antigen provided an indication of particular amino acid residues involved in antibody-epitope interactions. In particular, carbamylation perturbs lysine residues. This was clearly shown by MAb A55: previously shown to recognise a topographical epitope on the N-terminal face of the CH2 domain incorporating lysine 274 [4]. Evidently the antigenicity of MAb A57H was not affected by carbamylation. Recent interest has also focused on the ability of UV irradiation to induce oxidative stress in protein solutions where damage is mediated by oxygen free radicals. These highly reactive species degrade cysteine, proline and aromatic residues: tyrosine and tryptophan generating phenolic and fluorescent kyneurine derivatives Table 2. Epitope mapping of MAb A57H