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Volume 272, Number 5, Issue of January 31, 1997 pp. 2821-2827
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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Binding of the Brucella abortus Lipopolysaccharide O-chain Fragment to a Monoclonal Antibody
QUANTITATIVE ANALYSIS BY FLUORESCENCE QUENCHING AND POLARIZATION

(Received for publication, June 24, 1996, and in revised form, October 14, 1996)

From the Animal Diseases Research Institute, Nepean, Ontario, Canada K2H 8P9

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) µmol 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 × 10⁷ to 2.0 × 10⁷ M¹) 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.

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