Epitope mapping employing immobilized synthetic peptides. How specific is the reactivity of these peptides with antiserum raised against the parent protein?

Peptides synthesized onto polyethylene pins can be directly used to map the antigenic structure of proteins by ELISA (Geysen et al., 1987, J. Immunol. Methods 102, 259-274). The specificity of the reaction between anti-cytochrome c antibodies (IgG) and pin-bound hexapeptides of cytochrome c was tested using a competitive ELISA in which free and pin-bound peptides competed for the antibody. Competition was specific for many of the pin-bound peptides: only free peptides having the same sequence as the pin-bound peptides were able to compete for antibody-binding. However, non-specific reactivity was observed with pin-bound peptides whose sequence corresponded to the N terminal segment of cytochrome c. This segment is predicted to be particularly antigenic because of its high mobility and the nature of its amino acid sequence. In one case no competition by the free peptide could be observed even though the pin-bound peptide reacted strongly with anti-cytochrome c antibodies.