Re-evaluation of the concept of functional affinity as applied to bivalent antibody binding to cell surface antigens.
Although it is recognized that the bivalent binding of an antibody to a multivalent antigen cannot be characterized by true affinity, "functional affinity" is frequently determined. "Functional affinity" is calculated by the same methods used for true affinity, and is presumed to have similar biological significance. The data presented herein demonstrate, for IgG antibodies binding to cell surface antigens, that "functional affinity" does not adequately describe the interactions that occur. First, the dissociation rate was not first-order, but rather reflected heterogeneity of bound Ab. A minority of bound Ab (probably monovalently bound) dissociated relatively rapidly, while the majority dissociated very slowly, and, for most purposes, should be considered irreversibly bound. Although irreversible binding is incompatible with a state of equilibrium, the dissociation rate was greatly increased in the presence of free Ab, which may explain why equilibrium is reached in a Scatchard-type experiment. In addition, "functional affinity" was found to vary depending on trivial experimental conditions, such as the volume of incubation. Thus, antibodies do not possess a single "functional affinity", but rather a spectrum of "functional affinities", which are therefore of little value in comparing different antibodies and cannot be used to predict the extent of binding under other experimental conditions. Both of these discrepancies can be attributed to the effect of bivalent Ab binding. We conclude that "functional affinity" determination is inappropriate.
Ong
G L
GL
Center for Molecular Medicine and Immunology, Garden State Cancer Center, Newark, NJ 07103.
Mattes
M J
MJ
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000494:pharmacology; D000915:Antibody Affinity; D000954:Antigens, Surface / Q000276:immunology*; D004910:Erythrocyte Membrane / Q000276:immunology*; D006801:Humans; D051379:Mice
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface