Use of Natural or Selected Mutants and Variants for Epitope Mapping

Abstract

This brief chapter is less of a protocol and more of a reminder that valuable information on the location of epitopes can be obtained by studying antibody binding to naturally occurring variants of the antigen. These variants are usually antigens from different species or different isoforms of the antigen. Of course, amino acid sequences from different species must be available, but this now applies to a large and growing number of proteins on the data bases. Since these natural variants retain their function (e.g., enzyme activity), the amino acid changes are unlikely to have affected antibody binding by causing global changes in protein conformation. However, in the case of assembled epitopes, caution is still required before concluding that the altered amino acid is a “contact” residue within the epitope, especially if no supporting evidence is available. With linear epitopes, the conclusion can often be confirmed by using synthetic peptides. For globular proteins, mutations that have no effect on protein function alter surface amino acids more frequently than amino acids in the protein “core,” and it is these surface amino acids that are also involved in antibody binding. Consequently, the chances of an MAb displaying species specificity are rather high, even when the antigen is fairly highly conserved overall.