Measuring antibody affinity and performing immunoassay at the single molecule level.
Fluorescence correlation spectroscopy (FCS) enables direct observation of the translational diffusion of single fluorescent molecules in solution. When fluorescent hapten binds to antibody, analysis of FCS data yields the fractional amounts of free and bound hapten, allowing determination of the equilibrium binding constant. Equilibrium dissociation constants of anti-digoxin antibodies and corresponding fluorescein-labeled digoxigenin obtained by FCS and fluorescence polarization measurements are identical. It is also possible to follow a competitive displacement of the tracer from the antibody by unlabeled hapten using FCS in an immunoassay format. The fluorescence polarization immunoassay for vancomycin detection was used to test the FCS approach. Fitting of the FCS data for the molar fractions of free and bound fluorescein-labeled vancomycin yielded a calibration curve which could serve for determination of the vancomycin concentration in biological samples.
Drug Monitoring, Abbott Diagnostics Division, Abbott Park, IL 60064-6016, USA. sergey.tetin@abbott.com
Sergey Y
SY
Tetin
Kerry M
KM
Swift
Edmund D
ED
Matayoshi
D000818:Animals; D000915:Antibody Affinity / Q000276:immunology*; D000937:Antigen-Antibody Reactions; D001667:Binding, Competitive; D004076:Digoxigenin / Q000737:chemistry*; D004077:Digoxin / Q000276:immunology*; D016231:Fluorescence Polarization Immunoassay / Q000379:methods*; D005456:Fluorescent Dyes; D006241:Haptens; D007074:Immunoglobulin G / Q000032:analysis*; D011817:Rabbits; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D006241:Haptens; D007074:Immunoglobulin G; D004077:Digoxin; D004076:Digoxigenin