Sensitive titration microcalorimetric study of the binding of Salmonella O‐antigenic oligosaccharides by a monoclonal antibody

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Abstract
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 μmol 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 (ΔG°=− 31 kJ · mol−1; ΔH°=−21 kJ · mol−1 and −TΔS°−10 kJ · mol−1), while all four oligomers 2–5 showed a constant binding energy ΔG°=·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.