ANTI-ENZYME IMMUNITY 189 



and their antibodies. Horses were immunized against diphtheria cul- 

 ture fluid filtered through a Berkefeld candle. The immune horse sera 

 contained agglutinins against the diphtheria bacilli. A mixture of 

 diphtheria agglutinating sera (which contained no antitoxin) and 

 crude toxin gave a precipitate. After centrifuging the reaction mixture 

 the supernatant showed a marked drop in agglutinin content. This 

 same supernatant showed no measurable fall in the toxin content deter- 

 mined by skin tests on guinea pigs. Conversely, a plasma containing 

 both agglutinin and antitoxin after flocculation of antitoxin with toxin 

 showed no change in the agglutinin titer of the centrifuged super- 

 natant. Schmidt (1926) carried out similar tests as described above. 

 After treating a diphtheria agglutinating serum with crude toxin and 

 removing the floccules by centrifuging, he compared the toxicity of 

 the supernatant with a control sample of toxin treated with saline and 

 found no difference in their strength. These facts show that the 

 protein impurity present in the toxin solution reacts with its homolo- 

 gous antibody, and that the resulting precipitate does not adsorb 

 detectable amounts of the toxin present in the mixture. In an aggluti- 

 nating serum which also contains antitoxin the two reactions, toxin- 

 antitoxin and agglutinogen-agglutinin, take place independently. 



Marrack and Smith (1931) tested the mutual adsorbability of an 

 azo-globulin dye solution (prepared by linking purified horse serum 

 pseudo-globulin, or crystalline egg-albumin with diazotized atoxyl) 

 with diphtheria toxin-antitoxin floccules. Diphtheria toxin was mixed 

 with azo-globulin solution and then an equivalent amount of antitoxin 

 was added. The mixture was kept for three hours at 45 °C. and over- 

 night in the ice-chest. The centrifuged precipitate, dissolved in 0.01 

 N NaOH solution, was completely colorless. No azo-globulin was 

 therefore carried down with the toxin-antitoxin floccules. 



c. Failure of Protein-Anti-Protein Precipitates to Adsorb Non- 

 specific Colored Proteins. Marrack and Smith (1931a) mixed anti- 

 pseudoglobulin serum with azo-egg albumin solution; then one-third 

 the optimum proportion of horse pseudo-globulin was added. The mix- 

 ture was kept five hours at room temperature and in the ice-chest over- 

 night. The precipitate on dissolving in 0.01 N NaOH solution was 

 completely colorless. No dye azo-albumin was therefore carried down 

 by the pseudoglobulin-anti-pseudoglobulin precipitate. The absence 

 of azo-protein in the precipitate was determined spectroscopically with 



