Binding of the Brucella abortus lipopolysaccharide O-chain fragment to a monoclonal antibody. Quantitative analysis by fluorescence quenching and polarization.
An antigenic O-chain polysaccharide fragment derived from Brucella abortus lipopolysaccharide was labeled with 14.8 +/- 1.8 (n = 5) and 52.3 +/- 2.4 (n = 3) micromol of fluorescein/g of polysaccharide (designated FL1 and FL2, respectively) for use in investigating the binding of O-chain to a specific murine antibody YsT9 under equilibrium conditions. Upon binding to YsT9, the fluorescence of FL1 and FL2 was quenched 45-57% with no shift in the excitation and emission spectra, and polarization of fluorescence increased by 300-335%. With fluorescence quenching and polarization as sensitive signals for antibody-bound labeled O-chains, the equilibrium constants for binding of FL1, FL2, and unlabeled O-chain to YsT9 were determined to be within a similar order (1.5 x 10(7) to 2.0 x 10(7) M-1) using a nonlinear curve fitting approach rather than Scatchard analysis. These results indicated that covalent attachment of fluorescein groups to the O-chain did not influence the recognition of the YsT9-defined epitope by the antibody. The reversibility of the O-chain-antibody reaction was also demonstrated by showing a rapid depolarization of the labeled O-chain-antibody complex in the presence of unlabeled O-chain, suggesting that this displacement experiment could be exploited to quantify the Brucella polysaccharide antigen. The study described here provides a useful model for characterization of the complex formation between a carbohydrate-binding protein and a carbohydrate ligand and also for the design of a homogeneous assay system to quantitate antigens or antibodies of clinical interest.
Lin
M
M
Animal Diseases Research Institute, Nepean, Ontario, Canada K2H 8P9.
Nielsen
K
K
D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D000936:Antigen-Antibody Complex; D002003:Brucella abortus / Q000276:immunology*; D002850:Chromatography, Gel; D016650:Fluorescein-5-isothiocyanate; D005454:Fluorescence Polarization; D015151:Immunoblotting; D007700:Kinetics; D051379:Mice; D008956:Models, Chemical; D019081:O Antigens / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D019081:O Antigens; D016650:Fluorescein-5-isothiocyanate