ITC
Functional Application Areas
Application Note
Antibody Binding
Antibodies are produced by animals in response to the presence of a foreign material, such as proteins, and sugars, and small molecules (the antigen). Antibodies are an important component to the immune response. Antibody-antigen interactions are highly specific, and antibody structure and activity is actively studied in biotechnology. Since antibodies are so specific, they have been used as biotherapeutics against a variety of diseases.
Understanding how antibodies bind to their antigens, as well as how antibodies are formed into multi-domain proteins with variable and constant domains, is important in the development of biotherapeutics and vaccines.
Isothermal Titration Calorimetry (ITC) is a powerful analytical tool which measures the binding affinity and thermodynamics between any two biomolecules. ITC is considered the “gold standard” assay for binding.
ITC is vital in the study of multi-probe structure activity relationship (SAR) since it can detect contributions that affinity-only methods may miss. For example, the affinity measured by these methods may be similar for a wild-type and mutant antibodies binding to an antigen, but ITC can reveal differences in ΔH and ΔS that can describe the mechanism of action of binding. This information can validate in-silico modeling. ITC is also commonly used to validate other binding assays.
ITC is also used to characterize the regions of an antibody related to antigen specificity (i.e. a series of related antigens binding to the same site of an antibody, or performing site-directed mutagenesis of the antibody and see its effect on antigen binding), and studying the regions involved with antibody assembly.
References
Thermodynamic studies on the interaction of antibodies with beta-amyloid peptide.
Brockhaus M., Ganz P., Huber W., Bohrmann B., Loetscher H. R. and Seelig J.
J Phys. Chem B 111, 1238-1243 (2007)
Water molecules in the antibody-antigen interface of the structure of the Fab HyHEL-5-lysozyme complex at 1.7 A resolution: comparison with results from isothermal titration calorimetry.
Cohen G. H., Silverton E. W., Padlan E. A., Dyda F., Wibbenmeyer J. A., Willson R. C., and Davies D. R.
Acta Crystallogr D Biol Crystallogr 61, 628-633 (2005)
Measurement of protein interaction bioenergetics: application to structural variants of anti-sCD4 antibody.
Doyle M. L., Brigham-Burke M., Blackburn M. N., Brooks I. S., Smith T. M., Newman R., Reff M., Stafford W. F., III, Sweet R. W., Truneh A., Hensley P., and O'Shannessy D. J.
Methods Enzymol 323, 207-230 (2000)
ITC – Antibody Studies Reference List
Need more information? Contact us.
