ANALYSIS BY SURFACE-PLASMON RESONANCE OF THE INFLUENCE OF VALENCE ON THE LIGAND-BINDING AFFINITY AND KINETICS OF AN ANTICARBOHYDRATE ANTIBODY

The kinetics of ligand binding by Se155-4, an antibody specific for th e Salmonella serogroup B O-polysaccharide, were studied by surface pla smon resonance, Because trace amounts of oligomers in Fab and single-c hain antibody variable domain (scFv) preparations resulted in biphasic binding profiles that were difficult to analyze, all kinetic measurem ents were performed on purified monomeric fragments and, for certain m utant scFv, dimeric forms, Results obtained with monomeric forms indic ated that the relatively low affinity of the antibody was due to rapid dissociation (k(off) approximate to 0.25 s(-1)), Dimeric forms genera lly showed off-rates that were approximately 20-fold slower and a 5-fo ld 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 accessib ility of sites on the antigen always leads to some monovalent attachme nt, 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 d imer, whereas the on-rate was the same as that obtained with Fab and s cFv monomer.