272 INFECTION AND IMMUNITY 



quantity is mixed with varying dilutions of the unknown antitoxin. 7 

 Thus, given an antitoxin in which 300 to 400 units to the c.c. are 

 suspected, dilutions of 1:200, 1:250, 1:300, etc., are made. One 

 c.c. of each of these is mixed with the L + dose of the toxin, and 

 the mixtures are injected into guinea-pigs of about 250 grams. If 

 the guinea-pig receiving L + plus the 1 :250 dilution lives and the 

 one receiving L + plus the 1 :300 dilution dies in the given time, we 

 know that the unit sought must lie between these two values, and 

 further similar experiments will easily limit it more exactly. The 

 possibility of error in carrying out such measurement is much 

 diminished by the use of larger quantities of dilutions higher than 

 those given. Four c.c. is the volume usually injected. 



Since 1902, the production and sale of diphtheria antitoxin has 

 been regulated by law in the United States. From time to time, 

 antitoxin is bought in the open market and examined at the hygienic 

 laboratories of the United States Public Health and Marine Hospital 

 Service. Antitoxic serum which contains less than two hundred 

 units to each cubic centimeter is not permitted upon the market. 



In a previous section we have seen that Hiss and Atkinson 8 and 

 others have shown an increase in the globulin contents of blood 

 serum of immunized animals. It has been shown, furthermore, 

 that the precipitation of such serum with ammonium sulphate car- 

 ried down in the globulin precipitate all the antitoxic substances 

 contained in the serum. Upon a basis of globulin precipitation, 

 Gibson 9 has recently perfected a method of concentrating and puri- 

 fying diphtheria antitoxin for therapeutic use. This procedure, as 

 carried out at the New York Department of Health, is, in principle, 

 as follows: 



The serum, as taken from the horse, is heated to 56 C. for 

 twelvr hours. This converts about half of the pseudoglobulin into 

 euglobulin, the antitoxin remaining in the pseudoglobulin fraction. 10 

 It is then 11 precipitated with an equal volume of a saturated am- 

 monium sulphate solution. After two hours, the precipitate is caught 

 in a filter and redissolved in a quantity of water corresponding to 



T Ddnits, "Die Werthbem. der Heilsera, " in Kolle u. Wassormann. 



8 7/is\s and Atkinson, Jour. Exper. Med., v, 1900. 



8 Gibson, Jour, of Biol. Chem., i, 1906. 



111 Dr. Banzltaf, personal communication. 



11 Gibson and Collins, Jour, of Biol. Chem., iii, 1907. 



