1 40 IMMUNO-CATALYSIS 



substantiated by Pope and Healey (1938, 1939) with ultracentrifugal 

 experiments. Pope further showed that after treatment of antitoxic 

 pseudoglobulin with pepsin at pH 4.2, the antitoxic product is no 

 longer coagulable at 58 °C., whereas the inactive split component is 

 completely coagulable at this temperature. Peterman and Pappen- 

 heimer (1941) studied the physico-chemical properties of an antitoxic 

 pseudoglobulin preparation and found it to be homogeneous by sedi- 

 mentation, electrophoresis and diffusion criteria. Its molecular weight 

 (184,000) was nearly the same as that of normal horse pseudo- 

 globulin, but its mobility was different from those of any of the protein 

 components of normal serum. This antitoxic pseudoglobulin contained 

 86,000 antitoxic units per gram, and was only 43.5 per cent specifically 

 precipitable by toxin. After digestion with papain at pH 4.2 and the 

 coagulation of the split product at 58°C. the antitoxic material re- 

 maining in the supernatant was almost homogeneous. Its molecular 

 weight now was 113,000 and it contained 135,000 antitoxic units 

 per gram. 



In a comprehensive study Northrop (1942) reported the isolation of 

 crystalline diphtheria antitoxin from trypsin digested antitoxic material. 

 The crystalline antitoxin has a molecular weight of 90,000, which 

 is very nearly one-half of the molecular weight of the original antitoxic 

 horse serum pseudoglobulin, and smaller than the antitoxin obtained 

 by Peterman and Pappenheimer. The antitoxin of Northrop was 

 strictly homogeneous in the ultracentrifuge with a sedimentation con- 

 stant of 5.7X10~^^ (Rothen, 1942). The material showed only one 

 boundary in the electrophoresis cell at pH 7.3 or 3.0. It was 90 per cent 

 or more precipitable by diphtheria toxin and had about 700-900 anti- 

 toxic units per milligram protein nitrogen by the flocculation test and 

 about 700 units per milligram by the animal test. 



Immunologically the crystalline antitoxin was found to behave as 

 an antigenic entity distinct from the normal horse serum proteins 

 (Ten Broeck). The serum of a rabbit immunized against normal horse 

 serum gave a precipitate with 1/4000 ml. normal horse serum (con- 

 taining about 0.002 mg. of protein nitrogen) but gave no precipitate 

 with 1 ml. of a solution of purified antibody containing 1/10 mg. of 

 protein nitrogen. 



Guinea pigs sensitized by the subcutaneous injection of purified 

 antibody containing 0.003 mg. N gave a typical anaphylactic reaction 



