Analysis by surface plasmon resonance of the influence of valence on the ligand binding affinity and kinetics of an anti-carbohydrate antibody.
The kinetics of ligand binding by Se155-4, an antibody specific for the Salmonella serogroup B O-polysaccharide, were studied by surface plasmon resonance. Because trace amounts of oligomers in Fab and single-chain antibody variable domain (scFv) preparations resulted in biphasic binding profiles that were difficult to analyze, all kinetic measurements were performed on purified monomeric fragments and, for certain mutant scFv, dimeric forms. Results obtained with monomeric forms indicated that the relatively low affinity of the antibody was due to rapid dissociation (koff approximately 0.25 s-1). Dimeric forms generally showed off-rates that were approximately 20-fold slower and a 5-fold increase in association rate constants to approximately 2 x 10(5) M-1 s-1. Although the association phases for scFv dimers showed good curve fitting to a one component interaction model, the dissociation phases were biphasic, presumably because the availability and accessibility of sites on the antigen always leads to some monovalent attachment. The fast off-rate for dimers was the same as the monomer off-rate. Se155-4 IgG off-rates were very similar to those observed for scFv dimer, whereas the onrate was the same as that obtained with Fab and scFv monomer.
MacKenzie
C R
CR
Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada.
Hirama
T
T
Deng
S J
SJ
Bundle
D R
DR
Narang
S A
SA
Young
N M
NM
D000595:Amino Acid Sequence; D000918:Antibody Specificity; D000936:Antigen-Antibody Complex / Q000737:chemistry* / Q000378:metabolism; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000378:metabolism*; D007074:Immunoglobulin G / Q000737:chemistry / Q000378:metabolism*; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D046911:Macromolecular Substances; D008433:Mathematics; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D019081:O Antigens / Q000737:chemistry* / Q000276:immunology*; D009844:Oligosaccharides / Q000737:chemistry / Q000276:immunology; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012044:Regression Analysis; D012475:Salmonella / Q000276:immunology*; D013997:Time Factors
D000936:Antigen-Antibody Complex; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D007135:Immunoglobulin Variable Region; D008024:Ligands; D046911:Macromolecular Substances; D019081:O Antigens; D009844:Oligosaccharides; D011994:Recombinant Proteins