Heterogeneity of Antibody Sites in Their Relative Combining Affinities for Structurally Related Haptens

Summary The heterogeneity of combining sites in an antihapten antibody was investigated by competitive binding, which provides a method for determining whether different sites tend to combine more strongly with different, closely related haptens; i.e., whether the ratio of K’s for these haptens varies among the combining sites. Specifically, the effectiveness of varying concentrations of several haptens in displacing a reference hapten, radioactive p-iodobenzoate, from specifically purified anti-p-azobenzoate antibody was determined. Each hapten tested became relatively less effective as the degree of displacement increased, indicating heterogeneity of combining sites in their relative combining affinities for closely related substances. A correlation was noted between structural similarity of the competing hapten to the reference hapten and the magnitude of variation of the apparent relative K. The results showed that each of the competing haptens was capable of displacing all or nearly all of the reference hapten if a sufficiently high concentration of competitor was used. Thus, there are few sites capable of combining with p-iodobenzoate which cannot also accommodate the other isomeric iodobenzoates. In view of possible advantages of competitive binding as a means of estimating average equilibrium constants, the validity of the method was checked by comparison with direct binding, using 2 strongly bound haptens for the comparison, and with hapten inhibition of the precipitin reaction. Good agreement with the former method was observed. The agreement with hapten inhibition was only fair; differences were attributable to the nature of the reference substances displaced from antibody in the 2 methods (p-iodobenzoate vs. antigen).