Determination of kinetic constants for the interaction between a monoclonal antibody and peptides using surface plasmon resonance

Abstract

Differences in the affinity of a monoclonal antibody raised against the protein of tobacco mosaic virus for 15 related peptides (residues 134-146) carrying single-residue modifications were investigated using a novel biosensor technology (Pharmacia BIAcore). Analysis of the peptide-antibody interaction in real time allowed fast and reproducible measurements of both association and dissociation rate constants. Out of 15 mutant peptides analyzed, five were not recognized by the antibody at all, and seven were recognized as well as the wild-type peptide. For three of the peptides, the rate constants were different for the mutant and wild-type peptides. The pattern of residue recognition suggests that the epitope is formed by three residues (140, 143, and 144) in a helical conformation that mimics the structure in the protein. Even a minor modification of these residues totally abolishes recognition by the antibody. Modifications of adjacent residues result in small but significant differences in association and/or dissociation rate constants. One of the recognized residues is totally buried in the three-dimensional structure of TMV protein, suggesting that a structural rearrangement next to the helix occurs during protein-antibody interaction.

Induced Fit of an Epitope Peptide to a Monoclonal Antibody Probed with a Novel Parallel Surface Plasmon Resonance Assay

“…A previously characterized monoclonal antibody raised against tobacco mosaic virus protein was strongly suspected of binding to an epitope displayed on a contiguous face of an ␣-helix (24). In this particular case, further characterization by alanine scanning and SPR of the antigenic variant peptide corresponding to the original antigen epitope suggested that alanine substitution of a particular glutamic acid, presumably involved in a salt bridge with a vicinal arginine, may affect peptide recognition by stabilizing a conformation that is advantageous to antibody binding (3,24).…”

Molecular analysis of peptides from the GH loop of foot-and-mouth disease virus C-S30 using surface plasmon resonance: a role for kinetic rate constants

“…SPR biosensors allow fast and reliable screening of antigens and provide kinetic data on antigen-antibody interactions (Altschuh et al, 1992;Garland, 1996;Schuck, 1997). However, artifacts such as ligand site heterogeneity, ligand steric hindrance, mass-transport limitations, conformational change and non-specific binding have been identified and caution must be taken when considering the kinetic constants as absolute values (Schuck and Minton, 1996;O'Shannessy and Winzor, 1996;Hall et al, 1996).…”

Electrostatic Interactions in the Binding Pathway of a Transient Protein Complex Studied by NMR and Isothermal Titration Calorimetry

“…In typical stopped-flow binding measurements, the protein and ligand are rapidly mixed, and the extent of the reaction is monitored as a function of time using techniques including spectroscopic absorbance (21), fluorescence (10,26,27), enzymatic activity (1,28,29), and surface plasmon resonance (30,31). These techniques are limited by the requirement that the lifetimes of the free and bound states must be severalfold longer than the time required to completely mix the protein and ligand solutions; otherwise, the reaction proceeds nearly to completion before the start of the measurement period.…”