Determination of kinetic constants for the interaction between a monoclonal antibody and peptides using surface plasmon resonance.
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.
Altschuh
D
D
Institut de Biologie Moléculaire et Cellulaire, Laboratoire d'Immunochimie, Strasbourg, France.
Dubs
M C
MC
Weiss
E
E
Zeder-Lutz
G
G
Van Regenmortel
M H
MH
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry; D000936:Antigen-Antibody Complex; D004797:Enzyme-Linked Immunosorbent Assay; D007700:Kinetics; D008433:Mathematics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D014027:Tobacco Mosaic Virus; D014764:Viral Proteins / Q000737:chemistry / Q000276:immunology*; D014961:X-Ray Diffraction
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D014764:Viral Proteins