Development of recombinant B subunit of Shiga-like toxin 1 as a probe to detect carbohydrate ligands in immunochemical and flowcytometric application.

Measurement of carbohydrate binding activity of Escherichia coli Shiga-like toxin in a simple and quantitative way is an important step for evaluation of antibodies with therapeutic value and of effectiveness of vaccine treatment. We constructed a plasmid vector (pVT1-B5) to express carbohydrate binding (B) subunit of Shiga-like toxin 1 without expression of toxic (A) subunit, and established a simple method to purify the recombinant B subunit, which was then labeled with digoxigenin. The binding specificity of the digoxigenin-labeled B subunit for globotriaosylceramide was established by thin-layer chromatography immunostaining. We developed an enzyme-linked immunosorbent assay using immobilized glycolipids, demonstrating high sensitivity and clear-cut specificity of the assay. The digoxigenin-labeled B subunit was also readily applicable to the detection of cell surface carbohydrate ligands by flow cytometry.