2 HIGH-AFFINITY MONOCLONAL IGG2A ANTIBODIES WITH DIFFERING THERMODYNAMIC STABILITY DEMONSTRATE DISTINCT ANTIGEN-INDUCED CHANGES IN PROTEIN A-BINDING AFFINITY

Two IgG2a monoclonal antibodies (G10 and F11) are described which have similar affinity for human spleen ferritin and identical protein A-bi nding affinity. The two mAbs display changes in protein A-binding affi nity following binding of the antigen to its specific recognition site in the variable domains. However, while antigen-induced conformationa l 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 anti ferritin antibody (C5) high-affinity binding of the antigen does not c hange an inherently low ability to bind protein A. Differential scanni ng calorimetry revealed that the enthalpy and Gibb's free energy of th ermal 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 fol ding unit, which may be responsible for interactions between the antig en-binding site and the protein A-binding site. This study provides th e first demonstration that functionally significant interactions betwe en two recognition sites in antibodies of the same subclass can be mod ulated by subclass-independent structural variations associated with d ifferent thermodynamic stability. (C) 1998 Elsevier Science B.V. All r ights reserved.