Homogeneity and Heterogeneity of Toxins Produced by <i>Clostridium botulinum</i> Type C and D Strains

Five Clostridium botulinum strains were used in the present work, two of type C, C-Stockholm (C-ST) and C-CB19, and three of type D, D-South African (D-SA), D-1873, and D-CB16. The toxins, except for those of C-CB19 and D-CB16, were purified, and antisera were prepared in rabbits. To clarify the antigenicity of the toxins, neutralization and agar gel double-diffusion tests were performed. Anti-C-ST toxin serum neutralized two kinds of type C 1 toxin to a similar extent. Antisera against D-SA and D-1873 toxins, however, showed different neutralizing activity toward three type D toxins. Precipitin lines formed between D-SA and D-1873 toxins, and their antisera spurred to each other. From anti-D-SA toxin serum, two neutralizing fractions, one which neutralized D-SA, D-1873, and D-CB16 and one which neutralized only D-SA, were obtained. These results indicate that the antigenicities of D-SA and D-1873 toxins are not identical. Anti-C-ST toxin serum produced cross-neutralization on type D toxins, although the neutralization titer differed depending on the kind of toxin used. A precipitin line was formed with D-SA toxin, but not with D-1873; the developed line spurred to the C-ST toxin precipitin line. Two anti-D toxin sera also caused cross-neutralization on type C 1 toxins. However, the neutralizing activity of each serum to the same type C 1 toxin was different, and only anti-D-SA toxin serum developed a precipitin line with C-ST toxin which spurred to the D-SA toxin precipitin line. From anti-D-SA toxin serum, two different fractions capable of neutralizing C 1 and D toxins were obtained; one neutralized C-ST, C-CB19, and D-SA toxins, but not D-1873 and D-CB16, and the other neutralized all five toxins. There may be at least two common parts among C-ST, C-CB16, and D-SA toxin molecules. </jats:p

Four different monoclonal antibodies against type C1 toxin of Clostridium botulinum

Monoclonal antibodies against type C1 toxin produced by Clostridium botulinum type C strain Stockholm (C-ST) were prepared by fusion of BALB/c myeloma cells P3X63-Ag8, with spleen cells from the mice immunized by C-ST toxoid. About 5% of single-cell colonies in wells were found to produce antibodies against the toxin as determined by an enzyme-linked immunosorbent assay (ELISA). Four different hybridoma cell lines, no. 9, 12, 14, and 17, were established, cloned by limiting dilution, and intraperitoneally injected into mice to obtain the ascites fluids containing high-titered antibodies. The reactions of these antibodies to type C1 and D toxins of strains C-ST, D-1873, and D-South African (D-SA) were observed by both neutralization and ELISA tests. Three monoclonal antibodies, no. 9, 14, and 17, reacted with C-ST toxin, but only no. 17 highly neutralized the toxin. These antibodies did not react with type D toxins. On the contrary, no. 12 reacted with toxins of both C-ST and D-SA (but not of D-1873) and commonly neutralized these two toxins. This indicates that there is a common antigenic part between C-ST and D-SA toxin molecules which participates in the toxin-neutralizing reaction. The neutralization profiles of C-ST toxin by no. 12 and 17 antibodies were different in a time-to-death test of mice. The mechanisms of neutralization by no. 12 and 17 may be different.</jats:p

Four different monoclonal antibodies against type C1 toxin of Clostridium botulinum.

Monoclonal antibodies against type C1 toxin produced by Clostridium botulinum type C strain Stockholm (C-ST) were prepared by fusion of BALB/c myeloma cells P3X63-Ag8, with spleen cells from the mice immunized by C-ST toxoid. About 5% of single-cell colonies in wells were found to produce antibodies against the toxin as determined by an enzyme-linked immunosorbent assay (ELISA). Four different hybridoma cell lines, no. 9, 12, 14, and 17, were established, cloned by limiting dilution, and intraperitoneally injected into mice to obtain the ascites fluids containing high-titered antibodies. The reactions of these antibodies to type C1 and D toxins of strains C-ST, D-1873, and D-South African (D-SA) were observed by both neutralization and ELISA tests. Three monoclonal antibodies, no. 9, 14, and 17, reacted with C-ST toxin, but only no. 17 highly neutralized the toxin. These antibodies did not react with type D toxins. On the contrary, no. 12 reacted with toxins of both C-ST and D-SA (but not of D-1873) and commonly neutralized these two toxins. This indicates that there is a common antigenic part between C-ST and D-SA toxin molecules which participates in the toxin-neutralizing reaction. The neutralization profiles of C-ST toxin by no. 12 and 17 antibodies were different in a time-to-death test of mice. The mechanisms of neutralization by no. 12 and 17 may be different