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.
Ruan
Qiaoqiao
Q
Department of Biotechnology, Core Research and Development, Abbott Diagnostics Division, Abbott Laboratories, Abbott Park, IL 60064, USA.
Tetin
Sergey Y
SY
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology; D000937:Antigen-Antibody Reactions; D006801:Humans; D007118:Immunoassay / Q000379:methods; D051379:Mice; D008962:Models, Theoretical; D020097:Natriuretic Peptide, Brain / Q000276:immunology*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000276:immunology
D000911:Antibodies, Monoclonal; D019715:Tissue Inhibitor of Metalloproteinase-1; D020097:Natriuretic Peptide, Brain