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.
Miyashita
S
S
Department of Microbiology,University of Shizuoka School of Pharmaceutical Sciences, Shizuoka-shi, Japan.
Matsuura
Y
Y
Miyamoto
D
D
Suzuki
Y
Y
Imai
Y
Y
D002241:Carbohydrates / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004926:Escherichia coli; D005434:Flow Cytometry / Q000379:methods; D006017:Glycolipids / Q000032:analysis / Q000378:metabolism; D006801:Humans; D008024:Ligands; D015335:Molecular Probes; D011994:Recombinant Proteins / Q000378:metabolism; D022622:Shiga Toxin 1 / Q000302:isolation & purification / Q000378:metabolism*; D014281:Trihexosylceramides / Q000032:analysis; D014407:Tumor Cells, Cultured
D002241:Carbohydrates; D006017:Glycolipids; D008024:Ligands; D015335:Molecular Probes; D011994:Recombinant Proteins; D022622:Shiga Toxin 1; D014281:Trihexosylceramides; C018549:globotriaosylceramide