HyHEL-8, HH10 and HH26, respectively) are murine monoclonal IgG 1 antibodies which share over 90% variable-region amino acid sequence identity and recognize identical structurally-characterized epitopes on hen egg white lysozyme (HEL). Previous immunochemical and surface plasmon resonance-based studies have shown that these antibodies differ widely in their tolerance of mutations in the epitope. While HH8 is the most cross-reactive, HH26 is rigidified by a more-extensive network of intramolecular salt links, and is highly specific, with both association and dissociation rates strongly affected by epitope mutations. HH10 is of intermediate specificity, and epitope mutations produce changes primarily in the dissociation rate. Calorimetric characterization of the association energetics of these three antibodies with the native antigen HEL and with Japanese quail egg white lysozyme (JQL), a naturally occurring avian variant, shows that the energetics of interaction correlate with cross-reactivity and specificity. These results suggest that the greater cross-reactivity of HH8 may be mediated by a combination of conformational flexibility and less specific intermolecular interactions. Thermodynamic calculations suggest that upon association HH8 incurs the largest configurational entropic penalty and also the smallest loss of enthalpic driving force with variant antigen. Much smaller structural perturbations are expected in the formation of the less flexible HH26 complex, and the large loss of enthalpic driving force observed with variant antigen reflects its specificity. The observed thermodynamic parameters correlate well with the observed functional behavior of the antibodies and illustrate fundamental differences in thermodynamic characteristics between cross-reactive and specific molecular recognition. KeywordsProtein-protein recognition; antibody-antigen complexes; cross-reactivity and specificity; titration calorimetry; affinity maturation; thermodynamicsThe association of antibodies with antigens is a critical component of immune function, and the processes of recognition and association are underlying features of all protein-protein interactions. The immune system is, however, unique in the occurrence of both highly specific and non-specific (cross-reactive and poly-reactive) interactions involving different types of Phone: (713) Fax: (713) antibodies. An understanding of antibody-antigen association is of growing importance for engineering of antibodies for therapeutic and diagnostic applications. Several antibody-antigen complexes have been extensively characterized not only for their immunological and clinical interest, but also as model systems to elucidate the general principles of protein-protein interactions (1-3). Antibodies recognizing hen egg white lysozyme (HEL) have often been used, with the majority of these studies addressing molecular, thermodynamic and kinetic features of antibodies with high specificity. There are significantly fewer reports addressing the molecular basis of re...