Distinct antigenic specificities of alanine peptide determinants attached to protein carriers via terminal amino or carboxyl groups.

Abstract

L‐Alanine chains, serving as haptenic groups were coupled to proteins in opposite directions. There was no cross‐precipitation reaction between “anti‐amino‐alanyl” antibodies (obtained by immunization with conjugates containing alanine chains with free α‐amino groups) and “anti‐alanyl‐carboxyl” antibodies (obtained by immunization with conjugates containing alanine chains with free α‐carboxyl groups). The properties of the combining sites of these antibodies were evaluated by means of the hapten‐inihibtion method. For the inhibition of the precipitin reaction, series of free peptides and peptide derivatives (blocked in th amino or carboxyl function) composed of L‐ and D‐alanine residues were employed. The results led to the conclusion that the overall properties of the combining sites in both systems, but not their specificity, were quite similar. The size of the combining site of “anti‐amino‐alanyl” antibodies is such as to accommodate a tripeptide, whereas that of “anti‐analy‐carboxyl” antibodies is such as to accommodate tri‐to a tetrapeptide. In both systems the most exposed portion of the antigeic determinant is immunodominant. Thus, “anti‐amino‐alanyl” antibodies bind very poorly peptides in which the amino terminal end was altered (e.g., by replacing the N‐terminal L‐alanine residue by a D‐residue, or by N‐acetylation), but will combine very effectively with amides of L‐alanine peptides. Correspondingly, “anti‐alanyl‐carboxyl” antibodies react very poorly with alanine peptides in which the aminio terminal end was altered, e.g., they do not bind amides of L‐alanine peptides. Both types of antibodies can react with free L‐alanine peptides, but in their combining sites these peptides are probably bond in oppoiste direction. Thus, antibodies with distinctly different specificities can be elicited against L‐alanine peptide determinants, depending on their mode of attachment in the immunogen.It is also shown that more precise information on the combining sites is obtained by comparing pairs of haptens of equal size which differ from one another in a particular feature, rather than from studying the effect of elongation in a homologous series of haptens.