Pathogenesis of Shigella diarrhea. IX. Simplified high yield purification of Shigella toxin and characterization of subunit composition and function by the use of subunit-specific monoclonal and polyclonal antibodies.
A simple purification scheme for shigella cytotoxin was devised, resulting in high yields (approximately 50%) and a 1,300-fold increase in specific activity compared with the initial crude bacterial cell lysate. The purified toxin was enterotoxic in ligated rabbit ileal loops and neurotoxic when injected into the peritoneal cavity of mice. Measurement of specific activity of cytotoxin and enterotoxin demonstrated that these two toxicities copurify during the fractionation procedure. On sodium dodecyl sulfate gel electrophoresis, the toxin migrated as two polypeptide subunits, an A subunit of 32,000 mol wt and a B subunit of 6,500 mol wt. Chemical cross-linking experiments demonstrate that the toxin is a complex consisting of one A and five B subunits with a molecular weight of 64,000. Polyclonal rabbit anti-toxin and anti-subunit B antisera were produced as well as subunit-specific mouse monoclonal antibodies. All antibodies preincubated with toxin neutralized cytotoxic effects in HeLa cell monolayers. In contrast, only A subunit-specific antibodies were able to neutralize toxin prebound to the HeLa cell surface. Antibody to the B subunit also inhibited binding of 125I-labeled toxin to these cells by 94% or more. These data demonstrate that the B subunit is involved in shigella toxin binding to the cell surface.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D001427:Bacterial Toxins / Q000302:isolation & purification*; D004405:Dysentery, Bacillary / Q000503:physiopathology*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D046911:Macromolecular Substances; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D011817:Rabbits; D022603:Shiga Toxins; D012762:Shigella dysenteriae
D000596:Amino Acids; D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D001427:Bacterial Toxins; D046911:Macromolecular Substances; D022603:Shiga Toxins