Neutralization of hemolytic and mouse lethal activities of C. perfringens alpha-toxin need simultaneous blockade of two epitopes by monoclonal antibodies.

Three murine monoclonal antibodies (MAbs 3B4, 1E8, 1F9) were produced by fusion of X63-Ag8.653 myeloma cells and splenocytes of mice immunized with glutaraldehyde-inactivated alpha-toxin of Clostridium perfringens. All MAbs belonged to the immunoglobulin G (IgG) class and possessed a kappa light chain. All the MAbs were specific for alpha-toxin of C. perfringens as demonstrated by immunoblotting experiments performed with culture supernatants of C. perfringens, C. bifermentans, C. sordellii, and Bacillus cereus. Competition analysis in an ELISA revealed that the MAbs recognized different epitopes on the alpha-toxin molecule. In an immunoblot assay based on a recombinant protein expressed in Escherichia coli, the binding site of MAb 1E8 but not those of MAbs 3B4 and 1 F9 were mapped to the COOH-terminal fragment of alpha-toxin (aa 248-370). To prove the neutralizing potential of the MAbs, alpha-toxin was preincubated with MAbs and subsequently tested for its lecithinase activity in an egg yolk diffusion turbidity (EYDT) assay, its hemolytic activity in a hemolysis test, and its lethal effect on mice after intraperitoneally administration. When the MAbs were tested individually, neutralization was only seen in the EYDT assay, where MAb 3B4 completely abolished the lecithinase activity of alpha toxin. However, when MAbs 3B4 and 1 E8 were used in combination, they acted synergistically and inhibited the lysis of rabbit erythrocytes in vitro. The same mixture of MAbs was also able to completely neutralize the lethal effect of three LD50 of alpha-toxin on Balb/c mice. Our results suggest that the alpha-toxin molecule contains several domains which are differently involved in the various activities of the toxin. We conclude that the hemolytic domain(s) of alpha-toxin is (are) identical with or very closely located to the domain(s) that cause the mouse lethal effect. The lecithinase activity may be involved in the mechanisms of hemolysis and mouse lethality but appears not to be the only determinant.