Preparation, analysis and antibody binding studies of simultaneously synthesized soluble and cellulose-bound HIV-1 p24 peptide epitope libraries

Summary Epitope libraries of the HIV-1 p24 epitope GATPQDLNTM, recognized by the murine monoclonal antibody CB 4-1, were prepared by simultaneous synthesis on single resin supports (solution phase library) and on a continuous cellulose membrane support (solid phase-bound library) Each position of the epitope was replaced by 19 l-amino acids (cysteine omitted) in the soluble library or by 20 l-amino acids in the cellulose-bound library. The soluble library was synthesized by simultaneously incorporating equimolar amino acid mixtures at each position of the epitope or by synthesizing single epitope analogues. The peptide mixtures were subsequently analyzed by HPLC, CZE and MALDI-TOF mass spectrometry. Double coupling of equimolar amino acid mixtures of either 0.8 equiv (coupling at epitope positions 6–10) or 1.5 equiv (coupling at epitope positions 1–5) resulted in approximately equimolar incorporation of all single components of the mixture. The mixtures were then separated by preparative HPLC, and the peptides or peptide mixtures of single fractions were isolated and analyzed for binding CB 4-1. The results were compared with those obtained from antibody binding studies using the cellulose-bound epitope library. The affinity constants of the soluble peptide variants qualitatively correlated with the binding of CB 4-1 to single cellulose-bound analogues. Both approaches allowed the rapid identification of key residues in antibody binding, thus giving insight into the molecular nature of this antibody-peptide interaction.