Antibody affinity maturation plays an essential role during an immune response, resulting in the generation of highly matured antibodies. It is well known that the antigen-binding mechanism varies during the maturation process. The well-shaped form for antigen-binding is often favored in matured antibodies and enables them to bind to antigens with high affinity. However, molecular insight into the correlation between antigen-binding and affinity maturation is limited. This information could be useful for the elucidation of an immunological response. It has been previously shown that at least two antibody types are secreted after immunization with (4-hydroxy-3-nitrophenyl)acetyl (NP). One of the antibody types appeared during an early stage of the immune response, while the second type appeared at a late stage of immunization. A key residue of these antibodies is located at position 95 on the heavy chain; the former type has Tyr (Tyr_H95-type) and the latter type has Gly (Gly_H95-type). Although Fv domains of these antibodies were encoded by the same genes present on variable heavy and light chains, Gly_H95-type antibodies have ~10-100 fold higher binding affinity to NP than those of Tyr_H95-type antibodies. We examined the biophysical properties of single-chain Fvs (scFvs) of Tyr_H95- and Gly_H95-type antibodies. Antigen-binding and thermal stabilities of scFvs were evaluated using isothermal titration calorimetry and differential scanning calorimetry, respectively. Thermodynamic analysis enabled us to discuss affinity maturation and adaptive dynamic nature of the immune response.