Sensitive titration microcalorimetric study of the binding of Salmonella O-antigenic oligosaccharides by a monoclonal antibody.
The binding of several oligosaccharide haptens by a monoclonal antibody, Se155-4, specific for Salmonella serogroup B O-antigen was studied by titration microcalorimetry. In the software developed by Wiseman et al. [Wiseman, T., Williston, S. & Brandts, J.F. (1989) Anal. Biochem. 17, 131-137] the number of binding sites/macromolecule is one of the optional regression parameters in the non-linear least-squares analysis of the calorimetric data. Instead, an approach was adopted in which the concentration of binding sites was treated as a regression parameter, obviating the requirement for precise values of antibody absorption coefficients and minimizing effects due to partially inactive antibody preparations. Furthermore, performing the least-squares analysis in two steps, first using a differential heat mode and then an integral heat mode, was shown to yield the most accurate results. The technique gave accurate results using not more than 1-2 mumol ligand and less than 7 mg antibody. Haptens 2-5 were oligomers of the O-antigenic repeating unit varying in chain length by 2-5 repeating units and a trisaccharide glycoside 1, which filled the binding site. The latter hapten exhibited a favourable entropy contribution to binding (delta Go = -31 kJ.mol-1; delta Ho = -21 kJ.mol-1 and -T delta So -10 kJ.mol-1), while all four oligomers 2-5 showed a constant binding energy delta Go = -33 kJ.mol-1, composed of increasingly stronger enthalpy forces compensated by an increasingly unfavourable entropy contribution. These observations are compared with results from enzyme immunoassays and a high-resolution crystal structure for the dodecasaccharide 3 bound to the Fab derived from Se155-4.
Sigurskjold
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BW
Institute for Biological Sciences, National Research Council of Canada, Ottawa.
Altman
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D000911:Antibodies, Monoclonal / Q000276:immunology*; D000942:Antigens, Bacterial / Q000276:immunology*; D001666:Binding Sites, Antibody; D002151:Calorimetry / Q000379:methods; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000276:immunology*; D011135:Polysaccharides, Bacterial / Q000276:immunology*; D012044:Regression Analysis; D012475:Salmonella / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D000939:Epitopes; D008024:Ligands; D009844:Oligosaccharides; D011135:Polysaccharides, Bacterial