A monoclonal antibody (BALB/c mouse) with specificity for a neutralizing epitope on the epsilon-toxin produced by Clostridium perfringens type D was used to raise anti-idiotypic antibodies (anti-Id) in different strains of mice and rabbits. These were purified and used in cross-immunization studies to induce anti-(antiidiotype). All strains of mice and rabbits immunized with BALB/c-derived anti-Id showed a high-titer antibody response directed towards the active site of the toxin. This protected the animals against toxin challenge and against an oral dose of the vegetative organisms. Animals immunized with other anti-Id preparations showed no specific antibody response and were not protected. Guinea pig peritoneal macrophages have a cell surface receptor for the toxin, and incubation of these cells with BALB/c anti-Id allowed them to survive toxin challenge, indicating that occupation of the receptors by the anti-Id prevented binding by the toxin. In conclusion, we have shown that an internal-image anti-Id preparation will induce protective immunity in syngeneic and xenogeneic animals and furthermore that immunity to a single epitope on the exotoxin is sufficient to protect against the toxin and clinical sequelae evoked by the disease-causing organism itself.

A panel of monoclonal antibodies specific for the Clostridium perfringens alpha-toxin was produced by the fusion of X63.Ag8-653 cells with splenocytes from mice immunized either intrasplenically or intraperitoneally with an alpha-toxoid. The toxin-binding activity of each monoclonal antibody was evaluated. The monoclonal antibodies were also screened for their toxin-neutralizing potential in vitro, as determined by the inhibition of phospholipase C and hemolytic activities. In vivo inhibition of toxicity was assessed by the survival of mice challenged with preincubated alpha-toxin-antibody mixtures. Only one monoclonal antibody (3A4D10) was protective in vivo and neutralizing in both in vitro assays. Since 3A4D10 could inhibit both activities, the evidence suggests that these are colocated in the same area of the toxin molecule. This paper identifies a significant continuous linear binding region for 3A4D10 at positions 193 to 198 in the primary amino acid sequence of alpha-toxin.

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