Two high-affinity monoclonal IgG2a antibodies with differing thermodynamic stability demonstrate distinct antigen-induced changes in protein A-binding affinity

• Published in: Journal of immunological methods Vol. 217; no. 1; pp. 131 - 141
• Main Authors: Kravchuk, Zinaida I., Chumanevich, Alexander A., Vlasov, Alexander P., Martsev, Sergey P.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 01.08.1998; Elsevier
• Subjects: Affinity; Animals; Antibodies, Monoclonal - chemistry; Antibodies, Monoclonal - immunology; Antibodies, Monoclonal - metabolism; Antibody Affinity; Antigen-Antibody Reactions; Antigen-antibody reactions, antigen-antibody complexes, antibody-complement and others. Study of affinity. Antigen presentation; Binding, Competitive; Biological and medical sciences; Calorimetry, Differential Scanning; Differential scanning calorimetry; Ferritins - immunology; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Humans; IgG2a antibodies; Immunoglobulin G - chemistry; Immunoglobulin G - immunology; Immunoglobulin G - metabolism; Mice; Molecular immunology; Protein A; Protein Conformation; Protein Denaturation; Spleen - chemistry; Stability; Staphylococcal Protein A - metabolism; Thermodynamics; Differential scanning calorimetry; Δ G d: Gibb's free energy of denaturation; IgG2a antibodies; Stability; Δ H d: overall transition enthalpy of thermal unfolding; T m: midpoint transition temperature of thermal unfolding of a `calorimetric domain; Affinity; CDR: Complementarity-determining region; Protein A; Ligand binding; Biological fixation; Immunoglobulins; Conformational dynamics; Molecular interaction; Thermodynamic stability; Monoclonal antibody; IgG2; Induced conformation
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/S0022-1759(98)00110-0

Two IgG2a monoclonal antibodies (G10 and F11) are described which have similar affinity for human spleen ferritin and identical protein A-binding affinity. The two mAbs display changes in protein A-binding affinity following binding of the antigen to its specific recognition site in the variable domains. However, while antigen-induced conformational changes in G10 enhance its affinity to protein A, interaction of F11 with ferritin results in a significant decrease in protein A-binding affinity. In contrast to the IgG2a antibodies, using a mouse IgG1 antiferritin antibody (C5) high-affinity binding of the antigen does not change an inherently low ability to bind protein A. Differential scanning calorimetry revealed that the enthalpy and Gibb's free energy of thermal unfolding for G10 was 19% and 23% higher, respectively, than the corresponding parameters for F11. The lower structural energetics of F11 are associated with the absence of a calorimetrically revealed folding unit, which may be responsible for interactions between the antigen-binding site and the protein A-binding site. This study provides the first demonstration that functionally significant interactions between two recognition sites in antibodies of the same subclass can be modulated by subclass-independent structural variations associated with different thermodynamic stability.