On the validity of "functional affinity" determination for antibodies binding to cell surface antigens or other polyvalent antigens.
A widely used method of comparing different antibodies (Abs) is to determine their "functional affinities." This value is supposedly a constant that reflects the basic binding interaction between antibody and antigen and, if it is determined under standard conditions, allows the comparison of Abs used by different laboratories. However, I present here both theoretical and experimental evidence that, for Abs binding bivalently, functional affinity determinations seem to be invalid. Experimental data were obtained with erythrocyte targets, to eliminate interference due to the internalization or catabolism of bound Ab, but similar results have been obtained with tumor target cells. Several fundamental discrepancies between the theoretical expectations and the actual values obtained were demonstrated, all of which can be attributed to the effects of bivalent binding. One discrepancy is that the functional affinity determined did not equal the ratio of the rate constants for association and dissociation. A second is that the functional affinity was not a constant but, rather, depended on trivial experimental conditions, such as the volume of incubation. In addition, it has been recognized previously that functional affinity is affected strongly by the structure of a multivalent antigen and, in particular, by any change that makes it more or less likely that an Ab will bind bivalently. The value of other methods of comparing the avidity of different Abs is discussed. In many situations, Ab binding can be considered predominantly irreversible for practical purposes.
D000915:Antibody Affinity; D000937:Antigen-Antibody Reactions; D000954:Antigens, Surface / Q000276:immunology*; D004912:Erythrocytes / Q000276:immunology*; D006801:Humans
D000954:Antigens, Surface