Characterization and optimization of peptide arrays for the study of epitope-antibody interactions using surface plasmon resonance imaging.
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 microL) 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 non-interacting 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 x 10(8) M(-1) while variants made by the substitution of alanine for residues based on the binding motif had binding constants of 2.8 x 10(7), 5.0 x 10(6), and 2.0 x 10(6) M(-1).
Wegner
Greta J
GJ
Department of Chemistry, University of Wisconsin, Madison 53706, USA.
Lee
Hye Jin
HJ
Corn
Robert M
RM
D000906:Antibodies / Q000032:analysis*; D000939:Epitopes; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D010455:Peptides / Q000032:analysis* / Q000276:immunology; D020349:Surface Plasmon Resonance; D013816:Thermodynamics
D000906:Antibodies; D000939:Epitopes; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D010455:Peptides