title: Determining antibody stoichiometry using time-integrated fluorescence cumulant analysis.
abstract: We applied fluorescence fluctuation spectroscopy to resolve the binding heterogeneity of fluorescently labeled ligand derived from brain natriuretic peptide (BNP), a widely used diagnostic marker of heart failure, to a corresponding monoclonal antibody. This system includes three species: (1) free ligand molecules, (2) antibody with a single site occupied, and (3) antibody with both sites occupied. The method we used, time-integrated fluorescence cumulant analysis (TIFCA), utilizes cumulants of fluorescence fluctuations to resolve subpopulations of multiple fluorescent species freely diffusing in a solution. The values of the cumulants depend on the concentration, molecular brightness and diffusion time of the fluorescent molecules. The number of molecules in each species reflects the antibody affinity. We apply TIFCA to successfully establish the stoichiometry of the system, estimate affinity, and identify the presence of an inactive fraction of antigen in a single titration experiment.
authors.affiliation: Abbott Diagnostics Research, Abbott Park, Illinois 60064, USA.
authors.forename: Joseph P
authors.initials: JP
authors.lastname: Skinner
authors.forename: Bin
authors.initials: B
authors.lastname: Wu
authors.forename: Joachim D
authors.initials: JD
authors.lastname: Mueller
authors.forename: Sergey Y
authors.initials: SY
authors.lastname: Tetin
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology; D004058:Diffusion; D008024:Ligands; D020097:Natriuretic Peptide, Brain / Q000737:chemistry / Q000276:immunology; D011485:Protein Binding; D013050:Spectrometry, Fluorescence
chemical_list: D000911:Antibodies, Monoclonal; D008024:Ligands; D020097:Natriuretic Peptide, Brain