Characterisation of epitopes of pan-IgG/anti-G3m(u) and anti-Fc monoclonal antibodies
Introduction
Assay restriction is a common feature and problem of monoclonal antibodies (MAbs). In essence, MAbs may perform well in some assay systems but may be poor or ineffective in others [1], [2]. In addition, the specificity of a given reagent can also be determined by the assay system employed [3]. This phenomenon relates to the nature and display of the antigenic determinant, or epitope, and whether it is modified (or denatured) as a result of procedures used within an assay technique [3]. Consequently the characteristics of an antibody's target epitope in terms of location, composition of amino acids and accessibility are highly significant together with issues of antibody specificity and reactivity in the development of secure assay systems [4]. To this end, pepscan methodologies which employ a series of overlapping synthetic peptides of a given protein have provided an effective means of identifying key residues for antibody recognition and binding [5]. Overall, pepscan analysis employing peptides immobilised on polyethylene pins has been satisfactorily used to identify MAb reactivity to continuous epitopes [6]. However, a disadvantage of this particular methodology has been the uncertainty of peptide regeneration and thus pepscan using biotinylated peptides (attached to streptavidin coated plates) has also been employed [7]. In general, pepscan methodologies provide greater resolution of target residues compared with gross epitope mapping techniques such as enzyme degradation and cyanogen bromide cleavage, which allow the localisation of antibody reactivity to particular fragments [1].
Monoclonal reagents are widely used within the biomedical sciences and readily enable the standardisation of both reagent and assay technique because they are specific and can be generated in unlimited quantities in vitro [8]. Earlier studies [9], [10] have demonstrated two MAbs (PNF69C and PNF110A) with assay restriction profiles. Of interest is that both MAbs exhibit pan-IgG and anti-G3m(u) specificity in indirect ELISA/haemagglutination (HA) and capture ELISA/haemagglutination-inhibition (HAI), respectively. Both MAbs were derived from the same fusion but originated from different hybridoma cell lines. Both reagents may prove useful pan-IgG or anti-allotypic antibodies e.g. for clinical/forensic serology [11], although the target epitope(s) recognised by these antibodies remains unclear.
A previous report [12] has successfully employed biotinylated 15 mer overlapping peptides of IgG Fc to identify the target epitope of an in-house and commercially available pan-IgG monoclonal reagent, MAb A57H. This antibody recognised a peptide sequence 383-SNGQPENN-390 (Eu numbering, single amino acid code) that was found on all four IgG subclasses and straddled the fy1 strand and the b3 loop of the CH3 domain [13]. As an extension to this work, we have used these peptides to map potential epitopes of MAbs PNF69C and PNF110A and five other anti-IgG Fc MAbs (X1A11, WC2, G7C, JD312, 1A1) previously characterised with regard to subclass specificity. Furthermore by employing a software programme ‘PCImdad’ we have been able to demonstrate the surface topography and accessibility of potential IgG target epitope's identified through pepscan analysis.
Section snippets
Monoclonal antibodies
The generation and specificity of MAbs used in this study (PNF69C, PNF110A, X1A11, WC2, G7C, JD312 and 1A1) with regard to IgG subclass, allotypic (Gm) markers, deleted paraproteins and IgG fragments have previously been reported [9], [14], [15], [16]. These reagents have been characterised using a combination of techniques including haemagglutination (HA), haemagglutination-inhibition (HAI) and enzyme linked immunosorbent assay (ELISA). Five MAbs (TM15, ZB8, GOM1, ZG4 and RJ4) with reactivity
Western blotting
To ascertain the reactivity to potential linear epitopes within IgG Fc, seven MAbs (PNF69C, PNF110A, X1A11, WC2, G7C, JD312, 1A1) were evaluated by Western blotting. In this system, both reduced and non-reduced Fc and pFc′ fragments were used and their electrophoretic transfer to nitrocellulose validated by previously characterised antibodies. The data (Table 1) highlighted that MAbs PNF69C and PNF110A detected both reduced and non-reduced pFc′ (PER). MAbs X1A11 and 1A1 were reactive with Fc
Discussion
Epitope mapping using pepscan provides a useful adjunct to the characterisation of MAbs [4] and has been used in identifying epitopes on macromolecules including allergens and components of viruses [20], [21]. However, an important caveat of pepscan is that the technique is biased towards antibodies recognising linear, as opposed to conformational epitopes. Petrakou et al., [22] showed that only half of the MAbs investigated using overlapping peptides of MUC1 mucin yielded pepscan profiles. To
Acknowledgements
The authors would like to thank Mimotopes Pty Ltd. for the provision of synthetic peptides.
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