232 



Scientific Proceedings (121). 



when the cells are subsequently subjected to serum agglutination 

 but acid agglutination is not inhibited. 



A possibility which was kept in mind was that the effect with 

 antitoxin might be due to the presence of agglutinoids in antitoxic 

 serum. In an attempt to check this, agglutinating serum diluted 

 (1-10) was heated 75-80 0 C. for 1 hour and subsequently used in 

 the test; both with acid and serum agglutination there was no 

 inhibition. One might conclude from this experiment that the 

 agglutinins were destroyed by the high temperature and that no 

 agglutinoids were produced. Up to the present attempts to pro- 

 duce agglutinoids from agglutinins have not been successful. 



2. An emulsion of diphtheria organisms incubated with a 

 mixture of antitoxic serum and diphtheria toxin in suitable quan- 

 tities is subsequently agglutinated both by acid and by diphtheria- 

 agglutinating serum, whereas if the toxin is replaced by an equal 

 volume of broth the cells are not agglutinated. The conditions 

 for this experiment are limited by two factors: there must be 

 sufficient antitoxic serum to sensitize the cells, and an excess of 

 toxin. 



Diphtheria organisms sensitized with antitoxin are rendered 

 agglutinable by mixing with diphtheria toxin, centrifuging, wash- 

 ing with saline and re-suspending. 



Diphtheria toxin has this same neutralizing effect on diptheria- 

 agglutinating serum. From this one might conclude that diph- 

 theria toxin contains agglutinogens besides true toxin. 



3. In an attempt to apply the test quantitatively two general 

 methods were used: Organisms were sensitized with progressive 

 dilutions of antitoxic sera and the limit of inhibition read as the 

 end point; and mixtures of serum and toxin after standing 1 hour 

 at room temperature were added to sensitized cells directly, and 

 after dialyzing — it had been found that toxin dialyzes through 

 parchment paper — and the dialysate mixed with sensitized cells 

 which were subsequently tested for agglutinability. 



Neither method gave results which were uniformly consistent 

 with guinea-pig experiments. Using the first method some results 

 were obtained which were parallel to the guinea-pig tests, in other 

 cases the results were reversed, i.e., in some cases two sera which 



e o 



by Ehrlich's method gave, e.g., 300 A.U. and 150 A.U. per c.c. 



