To understand the mechanism of affinity maturation, we examined the antigen-antibody interactions between 4-hydroxy-3-nitrophenylacetyl (NP) caproic acid and the Fab fragments of three anti-NP antibodies, N1G9, 3B44, and 3B62, by isothermal titration calorimetry. The analyses have revealed that all of these interactions are mainly driven by negative changes in enthalpy. The enthalpy changes decreased linearly with temperature in the range of 25-45°C, producing negative changes in heat capacity. On the basis of the dependence of binding constants on the sodium chloride concentration, we have shown that, during the affinity maturation of the anti-NP antibody, the electrostatic effect does not significantly contribute to the increase in the binding affinity. We have found that, as the logarithm of the binding constants increases during the affinity maturation of the anti-NP antibody, the magnitudes of the corresponding enthalpy, heat capacity, and unitary entropy changes increase almost linearly. On the basis of this correlation, we have concluded that, during the affinity maturation of the anti-NP antibody, a better surface complementarity is attained in the specific complex in order to obtain a higher binding affinity.In affinity maturation of the immune response, the average affinity of the immunized serum generally increases with time after immunization (1). To investigate the relationship between the primary sequence diversity of antibodies and the progressive change in binding affinity, extensive analyses of antibodies have been carried out by using several haptens, 4-hydroxy-3-nitrophenylacetyl (NP) 1 (2-5), p-azophenylarsonate (6), phosphorylcholine (7), and 2-phenyl-5-oxazolone (8).A series of anti-NP mouse monoclonal IgG antibodies used in the present study were produced by the immune response of C57BL/6 mice against NP coupled to T cell-dependent carrier, chicken ␥-globulin (9, 10). The variable regions of the primary response anti-NP antibodies show low affinity for NP and carry few, if any, somatic mutations (2, 4), whereas those of the secondary response anti-NP antibodies usually exhibit increased affinity for NP and are somatically mutated (5). The secondary response antibodies are divided into two groups by carrying or lacking a somatic Trp 3 Leu exchange at position 33 in the variable region of the heavy chain (11,12). In the present study we compare N1G9, a primary response anti-NP antibody, with 3B44 and 3B62, which are secondary response anti-NP antibodies with and without Trp 3 Leu exchange, respectively. Thermodynamic aspect of antigen-antibody association is essential in order to understand the mechanism of the high affinity and specificity of antigen-antibody interaction. Thermodynamic parameters such as Gibbs free energy change, ⌬G, enthalpy change, ⌬H, entropy change, ⌬S, and heat capacity change, ⌬Cp, can provide useful information to identify fundamental forces involved in the antigen-antibody interaction. For instance, the magnitude of ⌬Cp is usually related to the contribution of the ...