Evaluation of peptide libraries: An iterative strategy to analyze the reactivity of peptide mixtures with antibodies

Peptide libraries corresponding to a presumed mixture of 50,625 tetrapeptides or 16,777,216 hexapeptides were each prepared in a single assembly by standard solid-phase peptide synthesis. By enzyme-linked immunosorbent assay, the tetrapeptide library was shown to inhibit the binding of an antiserum to FMRF amide with an FLRF capture antigen; the hexapeptide library was shown to inhibit the binding of a monoclonal antibody to a 28 amino acid peptide with the corresponding peptide capture antigen. An iterative strategy of variation was used to determine for each position in the tetra- or hexapeptides which amino acid contributed the most to activity. As a result we were able to logically select out of the tetrapeptide library the sequence FLRF and to select out of the hexapeptide library a sequence that differed from the apparent probable epitope but was twice as active. A single amino acid substitution in the logically derived sequence gave a peptide that was 35 times as active as the hexapeptide sequence in the original 28 amino acid peptide.