Evaluation of a pepscan approach to identify epitopes recognised by anti-hTSH monoclonal antibodies

In this study, several methodological aspects of the pepscan strategy have been investigated with the objective to delineate the amino acid sequences of peptide segments that form the epitopes of thyrotropin beta-subunit (TSH beta) recognised by monoclonal antibodies. Hitherto, the pepscan strategy has found application as an effective method to identify linear sequence re gions that constitute contiguous epitopes within the primary structure of s ome proteins. However, with heterodimeric glycoprotein hormones and their s ubunits such as TSH beta, as well as for many other globular proteins, the majority of the epitopes recognised by anti-protein antibodies will be deri ved from discontinuous segments that collectively form the epitope. In thes e cases the pepscan technique will only be able to identify individual segm ents of the overall discontinuous epitope site as linear peptides, some of which may interact with relatively low binding affinity. Consequently, addi tional attention must thus be given to the optimisation of the specific bin ding and detection conditions. Knowledge of the structures of these peptide segments can, however, provide a valuable basis to develop peptide structu res that more closely mimic the topographical features of the epitope in th e mature, folded protein. In an attempt to identify functional segments inv olved in the epitopes recognised by the anti-hTSH monoclonal antibodies, mA b279 and mAb299, the impact of various experimental conditions on the effic acy of the pepscan strategy has been investigated. The strategy involved th e synthesis of a series of overlapping pin-bound octapeptides with amino ac id sequences derived from the TSH beta-subunit. The ability of these pin-bo und octapeptides to bind to either mAb279 or mAb299 in ELISA-based assay wa s then determined under conditions involving different concentrations of th e primary and/or secondary antibodies, and changes in buffer composition, i ncubation times and washing procedures. The results of this study illustrat e some of the constraints and limitations of the pepscan technique when use d to delineate discontinuous epitopes of globular proteins, as well as prov iding insight into potential avenues to optimist and refine this method. (C ) 1999 Elsevier Science B.V. All rights reserved.