TOXINS AND ANTITOXINS 55 



that the intracutaneous injection of toxin into guinea-pigs leads to 

 localized necrosis in the course of forty-eight hours. The minimum 

 amount of toxin sufficient to produce necrosis can be determined, 

 the protective power of antitoxin determined, and subsequently 

 with a standard antitoxin any new toxin may be titrated after the 

 same general principles as described previously for antitoxin titra- 

 tion. By this method the presence of toxin in human blood may be 

 determined, inasmuch as normal human serum does not produce 

 necrosis upon intracutaneous injection in the guinea-pig. 

 Harriehausen and Wirth found that the serum from patients suffer- 

 ing with diphtheria produced necrosis owing to the presence of 

 toxin, and this was demonstrated in five cases for as long as thirty- 

 five days after the onset of the disease. By the use of a titrated 

 toxin the method may also be employed for determining the pres- 

 ence and amount of antitoxin in human blood. 



Active Immunization Against Diphtheria. For many years im- 

 munization to this disease was entirely in the form of passive im- 

 munization, practised by giving protective doses of antitoxin. The 

 antitoxin was given in doses of 500 to 1000 units and served to pro- 

 tect for a period of about three to six weeks. This was of special 

 importance in institutions and families exposed to the disease. The 

 disadvantages are the short period of immunity and the fact that 

 the patient may thereby become hypersensitive to any subsequent 

 injections of horse serum. Active immunity had been observed by 

 Park in guinea-pigs which had been used for the titration of anti- 

 toxin, and Park, in 1905, reported that horses treated with neutral- 

 ized mixtures of toxin and antitoxin had produced immune sera as 

 strong as 400 units per c.c. Theobald Smith suggested a similar 

 method of immunization in man, and in 1913 von Behring reported 

 the successful immunization of children and adults. The method 

 of immunization with toxin and antitoxin mixtures has now attained 

 a widespread use and is employed even as early as the fourth day of 

 life. Active immunity of this sort is demonstrable by the Schick 

 test in about ten days after treatment, and increases so that in the 

 eighth week about 80 per cent, of the treated individuals are immune, 

 by the twelfth week 96 per cent, are immune, and at the end of 

 four months 98 per cent, are immune. According to Park, the re- 

 maining 2 per cent, become immune if reinjected. The method of 

 immunization is to give three injections subcutaneously one 

 week apart. 



For the preparation of the mixture a ripe toxin is used and so diluted 

 that i. c.c. will contain 200 minimum lethal doses as tested against guinea- 

 pigs. This is slightly over-neutralized with antitoxin and the mixture should 

 cause no symptoms in guinea-pigs even when given in very large doses. As 

 indicated above, antitoxin may deteriorate in the moist state, and this must 

 be Q avoided in the toxin-antitoxin mixtures. If the mixtures are kept at about 

 21 C. the mixture remains good for at least one year, although it is prefer- 

 able to keep it at a lower temperature. The injection is given subcutaneously 

 in the arm at the insertion of the deltoid muscle. The immunity developed 

 following injection of this sort is against toxin, but vaccination against diph- 



