Characterization and Optimization of Peptide Arrays for the Study of Epitope−Antibody Interactions Using Surface Plasmon Resonance Imaging

Abstract

The characterization of peptide arrays on gold surfaces designed for the study of peptide−antibody interactions using surface plasmon resonance (SPR) imaging is described. A two-step process was used to prepare the peptide arrays: (i) a set of parallel microchannels was used to deliver chemical reagents to covalently attach peptide probes to the surface by a thiol−disulfide exchange reaction; (ii) a second microchannel with a wraparound design was used as a small-volume flow cell (5 μL) to introduce antibody solutions to the peptide surface. As a demonstration, the interactions of the FLAG epitope tag and monoclonal anti-FLAG M2 were monitored by SPR imaging using a peptide array. This peptide−antibody pair was studied because of its importance as a means to purify fusion proteins. The surface coverage of the FLAG peptide was precisely controlled by creating the peptide arrays on mixed monolayers of alkanethiols containing an amine-terminated surface and an inert alkanethiol. The mole fraction of peptide epitopes was also controlled by reacting solutions containing FLAG peptide and the noninteracting peptide HA or cysteine. By studying variants based on the FLAG binding motif, it was possible to distinguish peptides differing by a single amino acid substitution using SPR imaging. In addition, quantitative analysis of the signal was accomplished using the peptide array to simultaneously determine the binding constants of the antibody−peptide interactions for four peptides. The binding constant, K_ads, for the FLAG peptide was measured and found to be 1.5 × 10⁸ M^-1 while variants made by the substitution of alanine for residues based on the binding motif had binding constants of 2.8 × 10⁷, 5.0 × 10⁶, and 2.0 × 10⁶ M^-1.