350 IMMUNO-CATALYSIS 



It was suggested above that the proteolytic activity of the toxin might 

 spht or hydrolyze protein prior to the occurrence of the phenomenon 

 of opalescence. As found by Maschmann (1937) antitoxin neutralizes 

 the proteolytic activity, which effect might also be related to the in- 

 hibition of opalescence (hemolysis as well) by antitoxic sera. 



Following the detailed study of the phenomenon of opalescence by 

 MacFarlane, Oakley and Anderson (1941), van Heyningen (1941) 

 developed a refined technique to determine the concentration of a-toxin 

 in solutions by the measurement of the turbidity developed. He pre- 

 sented this method as a more accurate and economical method of 

 measuring the lethal, dermonecrotic and hemolytic properties of 

 toxin. He stated that the specific estimations of a-toxin by these 

 three methods are open to various objections, besides the time con- 

 sumed and the expense involved in carrying out the mouse experi- 

 ments. 



The substrate for toxin-enzyme action was prepared from the yolk 

 of a day-old egg. The turbidity developed was measured colorimetri- 

 cally. When amounts of toxin corresponding to 1-6 M.L.D. were in- 

 cubated for 15 minutes at 38° with 2 ml. of substrate solution, fairly 

 dense turbidities were produced, which fell outside the range of a 

 nephelometer, but which could be conveniently measured in an or- 

 dinary colorimeter. The degree of turbidity developed in a given time 

 was taken as a measure of the amount of toxin present. To stop the 

 reaction at the end of a desired period he added an equivalent amount 

 of the isotonic saline solution containing 2-3 antitoxin units per ml. 

 The activity of several samples of toxin could thus be determined 

 within an hour, with very satisfactory accuracy, and without resort- 

 ing to a large number of serial dilutions. It was stated to be particu- 

 larly useful in studying the toxin-antitoxin reaction since only one 

 tube need be used in the determination of the residual activity in a 

 toxin-antitoxin mixture.* 



h. Lecithinase Activity as a Measure of Biological Effects of 

 Toxin. MacFarlane and Knight (1941) found that the lecithinase 

 activity of the a-toxin is completely inhibited by the specific antitoxic 



*Crook (1942) reported the results of his study of the SeifiFert-Nagler reaction 

 (production of opalescence) with forty batches of toxin from thirteen different type 

 A strains and one type D strain of CI. welchii. He found that the correlation between 

 end point (Seiffert-Nagler reaction) and mouse M.L.D. was remarkably good through- 



