ANTI-ENZYME IMMUNITY 351 



serum. This inhibition was carried out as follows: Two ml. of diluted 

 toxin (1 to 2 lecithinase units) were allowed to react with a saline 

 dilution of the serum in a total volume of 3.8 ml. for 15 minutes at 

 room temperature; 0.2 ml. of 0.3 M CaCL, 1 ml. of borate buffer of 

 pH 7.1 and 1 ml. of 2.5 per cent lecithin were added and the amount 

 of hydrolysis in 15 minutes at 37° was estimated. A control without the 

 immune serum was likewise included. 



The results of numerous experiments showed that the hydrolysis of 

 lecithin by 1.9 lecithinase units of toxin was inhibited from 13 to 

 95 per cent by amounts of antiserum containing from 0.01 to 0.1 anti- 

 toxic units. The lecithinase activity was not significantly altered by 

 the addition of normal or diphtheria antitoxic horse sera in quantities 

 10 to 50 times greater than those of the CI. welchii antitoxic sera used. 

 When the lecithinase activity was measured in the presence of cal- 

 cium ion, a larger amount of antitoxin was required for the 100 per 

 cent neutralization of a given amount of toxin than in the absence of 

 calcium. To obtain complete neutralization of a given amount of 

 toxin, for example, it required only 0. 1 unit of antitoxin in the absence 

 of, and 0.19 unit in the presence of calcium ion. The effect of calcium 

 on the minimum hemolytic dose was similar. In the absence of cal- 

 cium, the minimum hemolytic dose of toxin was about three times as 

 much as in its presence. In this connection it is also interesting to 

 note that the toxoid, which is devoid of characteristic toxin activities 

 was likewise found to have lost the lecithinase activity. Unlike the 

 snake venom lecithinase which produced hemolytic lysolecithin, the 

 lecithinase of the a-toxin of CI. welchii hydrolyzes lecithin into non- 

 out. The correlation was even more accurate when the photo-electric method was 

 used. CI. oedematiens strains produced reactions as strong as CI. welchii filtrates made 

 under similar conditions. Crook stated that species such as CI. s-porogenes and CI. ter- 

 tium should be included among positives. In all cases where they were available, the 

 homologous antitoxins completely inhibited the reactions. Heterologous sera did not 

 neutralize the toxins in the Seiffert-Nagler reaction. His results also showed that the 

 reaction is due to the a-toxin of Glenny. The lipoid material separating during the 

 reaction is complex and consists of various types of lipoids and protein. 



The kinetics of the reaction in serum and egg-saline are characterized by a pro- 

 nounced induction period, and are typical of an enzyme or enzymes. 



Kass, et al. (1945) studied 94 strains of CI. welchii isolated from human and 

 animal feces and soil with respect to the interrelation of virulence and lecithinase 

 Cand hyaluronidase) production. Seventy per cent of all the strains produced leci- 

 thinase. Of the 46 per cent virulent strains only 17 per cent failed to produce 

 lecithinase. In a review, Reed (1943) discussed various aspects of Clostridia responsible 

 for gas gangrene. 



