756 TOXINS, TOXOIDS, AND ANTITOXINS 



streptococci. After about seven or eight months the serum contains considerable 

 antitoxin and has some antibacterial value. The Dicks' method of immunizing is by 

 the subcutaneous injections of increasing amounts of toxin from scarlatinal streptococ- 

 cus cultures which have been passed through a Berkefeld filter. Park and Williams 

 use a modification of Moser's method. They inject intravenously the killed centrifu- 

 gated twenty-four-hour culture from 50 cc. of broth. Two days later 50 cc. of a 

 six-day blood-broth culture which had been filtered through a Buchner funnel are 

 injected subcutaneously. The intravenous (dead culture) and subcutaneous (toxin 

 filtrate) doses are continued on alternate days and gradually increased. After eight 

 to nine months i mil of the serum usually contains sufi&cient antitoxin to neutralize 

 forty thousand S.T.D.'s, and 0.05 mil will protect mice against one hundred M.L.D.'s 

 of living streptococci. 



The antitoxin unit of scarlatinal streptococcus has been standardized and defined 

 as the amount of antitoxin which is required to neutralize fully one hundred S.T.D.'s 

 of a standardized scarlatinal toxin. 



PARTIAL PURIFICATION OF ANTITOXINS 



It has long been known that antitoxins are precipitated with the pseudoglobulin 

 fraction of horse serum. Gibson^ was the first to perfect a practical method of con- 

 centrating diphtheria antitoxin for therapeutic use. With his method it was only pos- 

 sible to concentrate about three times, due to the increase of the pseudoglobulin in an 

 antitoxin horse serum over the normal amount. It was thought by some workers that 

 this increase was a new-formed antitoxic pseudoglobulin, as it had all the then-known 

 characteristics of this protein. 



Gibson and Banzhaf-' showed that the increase of the pseudoglobulin is at its 

 height after one and one-half to three months, depending on the method of immuniza- 

 tion. The increase is from 40 to over 100 per cent. After several full bleedings there 

 is a gradual decrease of this protein, even though the unit value increases or remains 

 stationary. The writer^ was the first to point out that antitoxin could apparently be 

 dissociated from the pseudoglobulin without losing any of its activity. Possibly the 

 results to be described may be due not to dissociation but to a conversion of non- 

 antitoxin-bearing pseudoglobulins. Heating the antitoxic serum or plasma for six to 

 eight hours at 56° C., or three to four hours at 58° C., about 30 per cent of the pseudo- 

 globulin is converted into a euglobulin-like substance, i.e., one which has the salting- 

 out characteristics of the euglobulins. Heating for longer periods of time shows but 

 little further conversion of pseudoglobulin and considerable loss of antitoxin. It oc- 

 curred to the writer^ that the conversion curve was lessened, owing to the presence of 

 the greater amount of euglobulin in solution. Heating was then resorted to in the 

 presence of 30 per cent saturated ammonium sulphate solution to precipitate the 



'Dick, G. F., and Gladys H.: J. A.M. A., 82. 1246. 1924. 



' Gibson, R. B.: J. Biol. Chan., i, 161. 1906. 



3 Gibson, R. B., and Banzhaf, E. J.: /. Expcr. Med., 12, No. 3. 1910. 



"Banzhaf, E. J.: Proc. Soc. Expcr. Biol, b' Med., 5, 8. 1908; Bull. Johns Hopkins Hasp., 241. 

 106. 1911. 



5 Banzhaf, E. J.: Coll. Studies, Bureau of Lab. Depl. of Health, New York City, 7, 114. 1912. 



