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Bivalency and epitope specificity of a high-affinity IgG3 monoclonal antibody to the Streptococcus Group A carbohydrate antigen. Molecular modeling of a Fv fragment

2000; Elsevier BV; Volume: 324; Issue: 1 Linguagem: Inglês

10.1016/s0008-6215(99)00279-7

1873-426X

J. Bruce Pitner, Wayne F. Beyer, Thomas M. Venetta, Colleen M. Nycz, Michael Mitchell, Shannon L. Harris, Jose R. Mariño-Albernas, France‐Isabelle Auzanneau, Farzin Forooghian, Bernardine M. Pinto,

Escherichia coli research studies

The binding of Strep 9, a mouse monoclonal antibody (mAb) of the IgG3 subclass directed against the cell-wall polysaccharide of Group A Streptococcus (GAS), has been characterized. The intact antibody and proteolytic fragments of Strep 9 bind differently to GAS: the intact mAb and F(ab)′2 have greater affinity for the carbohydrate epitope than the monomeric Fab or F(ab)′. A mode of binding in which Strep 9 binds bivalently to portions of the polysaccharide on adjacent chains on GAS is proposed. A competitive ELISA protocol using a panel of carbohydrate inhibitors shows that the branched trisaccharide, β-d-GlcpNAc-(1→3)-[α-l-Rhap-(1→2)]-α-l-Rhap, and an extended surface are key components of the epitope recognized by Strep 9. Microcalorimetry measurements with the mAb and two synthetic haptens, a tetrasaccharide and a hexasaccharide, show enthalpy–entropy compensation as seen in other oligosaccharide–protein interactions. Molecular modeling of the antibody variable region by homology modeling techniques indicates a groove-shaped combining site that can readily accommodate extended surfaces. Visual docking of an oligosaccharide corresponding to the cell-wall polysaccharide into the site provides a putative model for the complex, in which a heptasaccharide unit occupies the site and the GlcpNAc residues of two adjacent branched trisaccharide units occupy binding pockets within the groove-shaped binding site.