Demonstration of the pH sensitive binding of multivalent carbohydrate ligands to immobilized Shiga-like toxin 1 B subunits.

The cytotoxic effects of Shiga-like toxins from enterohemorrhagic Escherichia coli O157:H7 depend on the recognition of carbohydrate determinants by B subunits. As a specific carbohydrate ligand, globotriaosylceramide has been characterized. We developed an alternative binding assay using multivalent carbohydrate ligands. We prepared globotriose-conjugated poly-lysine, and measured their binding to immobilized recombinant B subunits by an ELISA format. The signals representing ligand binding were dependent on the amount of immobilized B subunits as well as on the concentration of the ligands. The ligand binding activity was lost in an acidic environment, in which changes in the local conformation of the B subunits have been reported. Furthermore, pH dependent dissociation of the ligands from the B subunits was observed. We also demonstrate that antiserum from mice immunized with the B subunits specifically interferes with ligand binding. This suggests further potential for an assay to screen for blocking antibodies that could inhibit toxin internalization into host cells.