Applications of dual-color fluorescence cross-correlation spectroscopy in antibody binding studies |
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Authors: | Ruan Qiaoqiao Tetin Sergey Y |
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Affiliation: | Department of Biotechnology, Core Research and Development, Abbott Diagnostics Division, Abbott Laboratories, Abbott Park, IL 60064, USA. |
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Abstract: | 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. |
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Keywords: | Fluorescence correlation spectroscopy (FCS) Fluorescence fluctuation spectroscopy (FFS) Dual-color fluorescence cross-correlation spectroscopy (DC-FCCS) Protein interactions Antibody-antigen interactions |
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