78:889-896. production of the four major toxins, the alpha-, beta-, epsilon-, and iota-toxins (50). Epsilon-toxin is one of the toxins produced by type B and D strains (44). The epsilon-toxin can lead to a fatal illness (enterotoxemia) in a variety of livestock Asiaticoside animals, most frequently in sheep (50). Clinical indicators in intoxicated sheep may include colic, diarrhea, and numerous neurological symptoms. Postmortem analysis reveals widespread increases in vascular permeability with cerebral, cardiac, pulmonary, and kidney edema (52, 53). Experimental intoxication of mice and rats with epsilon-toxin causes a rapidly fatal illness and pathological changes similar to those seen in livestock (12, 13, 28, 46). The dose of epsilon-toxin required to kill 50% of mice has been estimated at between 65 and 110 ng per kg (27), Asiaticoside which indicates that epsilon-toxin is one of the most potent known bacterial protein toxins (14). Despite reports of epsilon-toxin-producing being isolated from humans, it is unclear whether or not epsilon-toxin causes illness in humans (15, 22, 26, 31, 34, 49). Due to the clear threat posed to livestock and the potential threat to human health, the epsilon-toxin is usually classified as a category B overlap select agent by the U.S. Department of Health and Human Services and the U.S. Department of Agriculture. To protect livestock from epsilon-toxin, both a vaccine (based on formalin-inactivated epsilon-toxin) and an equine-derived antitoxin are available. Due to the rapid progression of the disease among livestock animals, treatment is generally not possible or practical, and the emphasis is placed on prevention either by vaccination or by administration of antitoxin to unvaccinated animals in the event of an outbreak of enterotoxemia within a herd (2). Neither the antitoxin nor toxoid is usually approved for human use. Thus, both of the existing approaches to combat epsilon-toxin-mediated illness (approved for veterinary use) would be of limited value in response to exposure to weaponized epsilon-toxin. Alternative countermeasures are needed that inhibit the activity Rabbit polyclonal to UBE2V2 of the toxin. In this study, we characterized the inhibition of epsilon-toxin activity in vitro by two monoclonal antibodies, 4D7 and 5B7 (11, 18). These monoclonal antibodies neutralize the cytotoxic activity of epsilon-toxin in animal models of intoxication (1, 18, 46) and have also been used to study epsilon-toxin activity in vitro (11, 45, 46). Using an antibody competition enzyme-linked immunosorbent assay (ELISA), a peptide array, and a mutant recombinant epsilon-toxin, we mapped the epitope(s) recognized by the two neutralizing monoclonal antibodies. The epitope(s) recognized by the two antibodies overlaps the Asiaticoside putative membrane insertion domain name of the epsilon-toxin. MATERIALS AND METHODS Expression and purification of epsilon-prototoxin from type B strain ATCC 3626 (NCTC 13110, NCIMB 10691) was cultured anaerobically using the GasPak system (Becton Dickinson) in TGY medium (30 g per liter tryptone, 20 g per liter yeast extract, 5 g per liter dextrose, 0.5 g per liter cysteine) at 37C. An overnight culture was used to inoculate sterile TGY medium, and the resulting culture was incubated anaerobically for 7 h. The epsilon-prototoxin was purified by a combination of hydrophobic conversation and ion-exchange chromatography as previously described (29), with modifications. Proteins from filter-sterilized culture supernatant were precipitated with 70% ammonium sulfate, and the precipitated proteins were dissolved in 5 mM Tris (pH 7.5) and adjusted to 1 1 M ammonium sulfate. The protein sample then was applied to a phenyl-Sepharose column. The column was washed with 0.8 M ammonium sulfate in 5 mM.