The Complementarity Determining Regions (CDRs) of antibodies are assumed to account for the antigen recognition and binding and thus to contain also the antigen binding site. CDRs are typically discerned by searching for regions that are most different, in sequence or in structure, between different antibodies. Here, we show that ~20% of the antibody residues that actually bind the antigen fall outside the CDRs. However, virtually all antigen binding residues lie in regions of structural consensus across antibodies. Furthermore, we show that these regions of structural consensus which cover the antigen binding site are identifiable from the sequence of the antibody. Analyzing the predicted contribution of antigen binding residues to the stability of the antibody-antigen complex, we show that residues that fall outside of the traditionally defined CDRs are at least as important to antigen binding as residues within the CDRs, and in some cases, they are even more important energetically. Furthermore, antigen binding residues that fall outside of the structural consensus regions but within traditionally defined CDRs show a marginal energetic contribution to antigen binding. These findings allow for systematic and comprehensive identification of antigen binding sites, which can improve the understanding of antigenic interactions and may be useful in antibody engineering and B-cell epitope identification.

The production of antisera to specific proteins using as immunogens short, synthetic oligopeptides corresponding in sequence to regions of the proteins is analysed. Of 103 oligopeptides used for this purpose and reported in the literature before the end of 1983 all those corresponding to N or C terminal sequences produced antisera reacting with the complete protein. Of 69 oligopeptides corresponding to internal sequences only 71% were successfully used to prepare antisera. An analysis of these 69 oligopeptides showed that peptides of less than 10 amino acids were unlikely to produce useful antisera and that the more hydrophilic peptides were marginally more useful than those less hydrophilic.

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