Analysis of the specificity and thermodynamics of the interaction between low affinity antibodies and carbohydrate antigens using fluorescence spectroscopy

The purpose of this work has been to examine whether fluorescence spectroscopy can be used to investigate weak or transient binding between monoclonal antibodies and carbohydrate antigens. In earlier studies we have demonstrated that the three monoclonal antibodies 39.4 (IgG2b), 39.5 (IgG2b) and 61.1(IgG3) bind weakly to the glycosidic α(1-4) bond present in e.g. maltose and panose. In this study these antibodies showed an enhancement in the fluorescence intensity of tryptophan upon binding in solution to these two carbohydrate antigens. Using a structural analog to maltose, cellobiose, no fluorescence intensity change was induced. Dissociation constants for these antibodies at different temperatures (5-40 °C) were obtained in the range of 0.003-0.2 mM and they were in accordance with earlier data from studies on affinity chromatography and surface plasmon resonance. Almost a doubling of the dissociation constants was observed for every 10 °C increase in temperature, giving an exothermal reaction with standard enthalpy change of −51 kJ/mol, for the association between antibody and carbohydrate antigen. It was seen that the extra glycosyl ring in panose increased the affinity more than eight times for the monoclonal antibody 39.5. A standard entropy increase of 21%, probably due to hydrophobic effects, is introduced by the extra glycosyl ring, while the enthalpy stays unaffected. This direct fluorescence approach to measure the binding and thermodynamics of an interacting antigen-antibody pair is simple and accurate since measurements are performed in solution and no immobilization or fluorophore labeling of the components is required. Introduction of fluorescence techniques will be a useful complement to current procedures to measure interaction of antibody with antigen and in particular they will offer solutions to detect transiently binding antigens.