Applications of dual-color fluorescence cross-correlation spectroscopy in antibody binding studies

Two-photon dual-color fluorescence cross-correlation spectroscopy (DC–FCCS) was applied to study the binding interactions of monoclonal antibodies (mAbs) and protein antigens. We measured the binding constant of the interaction of a 32-amino acid brain natriuretic peptide (BNP) with a mAbs and demonstrated the utility of DC–FCCS in studies of antibody sandwiches, trimolecular formations, where two different antibodies bind the same antigen simultaneously. We also show the use of DC–FCCS for monitoring competitive displacement of the labeled antibody in antibody–antigen complexes and subsequent determination of the pertinent dissociation rate (off-rate). The off-rate measurements were performed for two mAbs toward tissue inhibitor 1 of metalloproteinases (TIMP-1). From a methodological perspective, selection of the best labeling protocols and careful optimization of the FCCS instrumentation are essential to achieve the highest cross-correlation signal. When working in vitro, it is practical to generate a complete binding curve using the normalized cross-correlation signal and then fit the experimental points to a binding model. DC–FCCS offers the sensitivity and all other advantages of a solution phase fluorescence-based technique. For systems containing proteins of a similar size that interact without substantial changes in the fluorescence intensity, DC–FCCS serves as a preferred means of measuring solution phase binding constants.