STUDIES ON IMMUNITY TO TOXINS OFCLOSTRIDIUM BOTULINUMVI

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Abstract
A method was developed for production and purification of Clostridium botulinum type D toxin for conversion to toxoid. Crude toxin was produced in a medium composed of corn steep liquor, glycerol and calcium carbonate. Toxicities of approximately 142 x 106 mouse intraperitoneal LD50 per ml were produced in cultures grown 19 days at 36[degree]C in intussuscepted cellophane tubing immersed in nutrient medium. The toxin was purified by a procedure that involved precipitation with alcohol in the cold followed by an extraction with calcium chloride solution and 2 additional alcohol precipitations. Specific activity of the purified toxin was approximately 500 x 106 LD50 per mg N. One of the most active preparations of toxin sedimented in the ultracentrifuge as a paucidisperse system in the pH range 3.8 to 6.7. The principal component, representing 80 to 90% qf the total concentration, had a sedimentation rate about equal to that of the type A toxin at pH 3.8. Toxin was converted to toxoid in the presence of 0.6% formalin at 33[degree]C, and after integration with aluminum phosphate was highly antigenic for mice, guinea pigs, and rabbits.