PRODUCTION AND TESTING OF ANTITOXINS 219 



measurement is much diminished by the use of larger quantities of dilu- 

 tions 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. Anti- 

 toxic 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 1 and 

 others have shown an increase in the globulin contents of blood serum 

 of immunized animals. It has been shown, furthermore, that the pre- 

 cipitation of such serum with ammonium sulphate carried down in the 

 globulin precipitate all the antitoxic substances contained in the serum. 

 Upon a basis of globulin precipitation, Gibson 2 has recently perfected a 

 method of concentrating and purifying diphtheria antitoxin for thera- 

 peutic use. This procedure, as carried out at the New York Depart- 

 ment of Health, is, in principle, as follows: 



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

 hours. This converts about half of the pseudoglobulin into euglobulin, 

 the antitoxin remaining in the pseudoglobulin fraction. 3 It is then 4 

 precipitated with an equal volume of a saturated ammonium sulphate 

 solution. After two hours, the precipitate is caught in a filter and 

 redissolved in a quantity of water corresponding to the original quantity 

 of serum. After filtration-, this solution is again precipitated with 

 saturated ammonium sulphate solution and the precipitate again fil- 

 tered off. The precipitate is then treated with a saturated solution of 

 sodium chloride of double the volume of the original serum. This is 

 allowed to stand for about twelve hours. At the end of this time the 

 antitoxin-containing globulin is in solution and is pipetted away from 

 the precipitate and filtered. This salt-solution extract is then pre- 

 cipitated with twenty-five hundredths per cent acetic acid. The re- 

 sulting precipitate of globulin is thoroughly dried by pressure between 

 filter papers and placed in a parchment dialyzer. Dialysis with run- 

 ning water is continued for seven to eight days, after neutralization with 

 sodium carbonate, in order to remove the sodium chloride. At the 

 end of this time, the globulin solution remaining in the dialyzer is fil- 



1 H iss and Atkinson, Jour. Exper. Med., v, 1900. 



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



3 Dr. Banzhaf, personal communication. 



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



