Aeromonas sobria produces hemolysin in a form activable with trypsin under defined cultural conditions. In immunoblotting analyses with the culture supernatant of A. sobria, the monoclonal antibody reacting specifically to Aeromonas hydrophila CA-11 hemolysin bound to the 53,000and 49,000-dalton bands before and after trypsinization, respectively. The monoclonal antibody reacting to A. hydrophila AH-1 hemolysin did not bind either band. A. sobria hemolysin is, therefore, related antigenically to CA-11 hemolysin, while the molecular weights before and after activation differ from those of A. hydrophila hemolysins, being 54,000 and 51,000, respectively. The hemolytic and enterotoxigenic activities of A. sobria hemolysin were both neutralized by the monoclonal antibody against CA-11 hemolysin. It seems, therefore, that the same site on A. sobria hemolysin is responsible for both biological activities.
The activity of hemolysin produced by Aeromonas hydrophila CA-li, isolated from an environmental source, was more sensitive to temperature than that of hemolysin produced by strain AH-1, isolated from a diarrheal case. CA-il hemolysin failed to elicit hemolysis below 10°C. Immunoblotting analyses showed that both hemolysins formed into oligomers in rabbit erythrocyte membrane even when no hemolysis occurred, suggesting that the binding and the subsequent oligomerization are temperature independent. Sodium salicylate inhibited lysis of rabbit erythrocytes by both hemolysins, but selected monosaccharides and oligosaccharides did not. Thin-layer immunostaining indicated that both hemolysins bound to phosphoglycerides with net negative charge but weakly to the ones with no net negative charge. Neither sphingomyelin nor lysophosphoglyceride reacted with the hemolysins, whereas the hemolysins bound to free fatty acids. These results suggest that the binding of either hemolysin to the membrane component, probably phospholipid, requires both negative charge of the polar head group and suitable hydrophobicity of the nonpolar tails.
Five monoclonal antibodies (MCA; E-8-2, 9-1, 11-2, 12-4, and 13-1) against Clostridiurn botulinum type E derivative toxin were prepared. Their ELISA titers were higher than or equivalent to that of conventional polyclonal antibody. Three of them (E-8-2, 12-4, and 13-1) possessed the neutralizing activity comparable to that of polyclonal antibody. The results of binding-competition experiments indicated that the monoclonal antibodies bound to different sites on the type E toxin molecule. Immunoblotting analyses demonstrated that E-8-2, 9-1, and 11-2 react to fragment I (heavy chain) of the toxin. By use of these monoclonal antibodies, it may be possible to scrutinize the structure-function relationship of botulinum toxins and cross reactions between type E and F toxins.
Summary. Two hybridoma cell lines that produce monoclonal antibodies against Aeromonas hydrophila haemolysin were established by fusion of myeloma and spleen cells obtained from a mouse immunised with haemolysin detoxified with tetranitromethane. Enzyme-linked immunosorbent assay (ELISA) showed that the two purified monoclonal antibodies, B7 and B1 1, recognised the same epitope on the haemolysin molecule. Antibody B7 neutralised the haemolytic and enterotoxic activities of the haemolysin. It is concluded that the same site on the haemolysin molecule is responsible for both haemolytic and enterotoxic activities.
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