ANTI-ENZYME IMMUNITY 305 



the controversy regarding the existence of anti-rennin antibody will be 

 analyzed below, we will begin with the description of Morgenroth's 

 results. 



Immunization of Goats with Rennin. The rennin preparation used 

 for the subcutaneous immunization of goats was prepared as follows: 

 A 1 per cent sodium chloride suspension of commercial Witte rennin 

 was shaken mechanically for a prolonged period. After centrifuging, 

 the insoluble sediment was discarded. The clear supernatant was 

 diluted with sterile water for injection. By a gradual increase of the 

 dose of rennin as much as 6.5 g. were injected subcutaneously into 

 each of two goats. One of the goats yielded a highly potent immune 

 serum; the other, not as good. A permanent stock-enzyme solution was 

 prepared which showed no decrease in activity during 1 8 months. 



After numerous preliminary experiments with respect to variations 

 of time, temperature and concentration, he developed a reliable method 

 for testing the clotting of milk with the enzyme solution. By allowing 

 the mixture of rennin and milk to react overnight at a temperature 

 of 0° to 8°C. before taking readings, consistent and reliable results 

 were obtained. His rennin solutions were carefully neutralized for 

 daily use to neutral red (pH 6.0) with a lactic acid solution. The 

 activity of the rennin solution in clotting cow's milk (about pH 

 6.58) was (by dry weight) 1 part in 3,000,000. 



The anti-rennin potency of the immune goat serum was determined 

 as follows: The cow's milk was treated with a volume of immune 

 serum to have a 2 per cent serum concentration in the mixture. To 

 each 5 ml. portion of this mixture increasing amounts of rennin solu- 

 tion were added at various intervals and parallel control tests were 

 carried out in the absence of immune serum to determine the relative 

 time of the clotting of the milk. In contrast to the control value of 

 1 : 3,000,000, in the immune reaction mixture containing 1 part of 

 rennin in 30,000 there was no clotting of the milk. When the concen- 

 tration of rennin reached 1 : 25,000 clotting was observed. These facts 

 showed that a 2 per cent immune serum neutralized more than 100 

 times the amount of rennin present in the control test. Another im- 

 mune serum tested identically was shown to neutralize 200 times the 

 amount of rennin present in the control reaction system. 



Morgenroth found that 0.05 ml. of diluted rennin solution was ca- 

 pable of coagulating 30 liters of milk. Thirty ml. of immune serum was 



