I050 ISOLATION OF SUBSTANCES WITH IMMUNE PROPERTIES 



teins, namely, a capacity to become selectively adsorbed upon homologous antigen. 

 Sheep erythrocytes, for example, added to chilled, homologous immune rabbit serum 

 not only were agglutinated and flocculated but also removed nearly all of the anti- 

 sheep immune substances from the serum. The adsorption of the immune substances 

 was demonstrated by centrifugating the agglutinated cells, washing them free of ad- 

 herent serum, resuspending them in physiological salt solution, and testing their 

 capacity to agglutinate untreated cell suspensions. The agglutinative capacity of the 

 artificial suspension resembled in every way a solution of the corresponding, whole, 

 immune serum. Moreover, a part of the adsorbed immune substances could be re- 

 covered from the sedimented cells by extraction with warm, isotonic salt solution. 



The recovery of the substances which become adsorbed from immune sera by 

 suspensions of homologous antigen was first accomplished by Hahn and Tromms- 

 dorf,' in 1900. Suspensions of bacteria were left in contact with homologous immune 

 serum until they had become completely agglutinated. The agglutinated sediments 

 were then separated, thoroughly washed with isotonic salt solution, and resuspended 

 in warm N/100 acid. Clear, agglutinin-containing extracts were obtained which gave 

 none of the accepted tests for the presence of protein. 



The extreme minuteness of the quantity of protein which may be associated with 

 a physiological effect is perhaps not generally appreciated. Egg albumin produces 

 anaphylactic sensitization in a quantity of less than 0.00005 mg.^ Purified diphtheria 

 toxin kills guinea pigs in quantities of less than 0.00007 ^g-^ The protein of the 

 spermatozoon produces specific fertilization and confers the male inheritance in a 

 quantity of 0.0000003 mg.i These quantities are many thousand times smaller than 

 may be recognized by any dependable chemical test for the presence of protein. The 

 substances carrying the immune property in Hahn and Trommsdorf's final extracts 

 were clearly associated with too minute an amount of material to permit of their 

 recognition and identification by any purely chemical method not requiring the con- 

 centration of prohibitively large volumes of extract. 



The yield of recovered immune substance obtained by the use of a modification 

 of their procedure is considerably augmented when the denaturation consequent to 

 long contact with warm N/ioo acid is avoided.^ Extraction with cold iV"/i,ooo acid, 

 dilute alkali, warm water, or warm isotonic salt and saccharose solutions is less 

 injurious.* Concentration of the extracts is effected by precipitation and re-solution 

 in a minimum quantity of dilute alkali or by evaporation in vacuo. Or the extracts 



' Hahn, M., and Trommsdorf, R.: Munclien. med. Wchnschr., 47,413. 1900. See also Lieber- 

 mann, L. von, and Fennyvessy, B. von: Centralhl.f. Bakteriol., 47, 274. 1908. 



^ Wells, H. G.: Chemical Aspects of Immunity, p. 28. Chemical Catalog Co., 1925. 



3 Locke, A., and Hirsch, E. F. : unpublished observation. 



4 Locke, A.: Proc. Inst. Med., 5, 316. Chicago, 1925. 



s Rondoni, P.: Ztschr.f. Immunitdtsforsch. u. e.xper. Therap., 7, 515. 1910. 



* Locke, A., and Hirsch, E. F.: /. Infect. Dis., 37, 449. 1925; Huntoon, F. M.: /. Immunol., 6, 

 117, 123, 185. 1921; U.S.P., No. 1484038. 1924; Ottenberg, R.: Proc. Soc. Exper. Biol. &" Med., 

 21, 14, 303. 1923-24. See also Gay, F. P., and Chickering, H. T.: /. Exper. Med., 21, 389. 1915; 

 Weinstein, I.: /. Immunol., 3, 17. 1918; Kosaki, M.; ibid., p. 109. 1918; Furuhata, T.: Japan 

 Med. World, i, i. 1921; Uchida, S.: /. Infect. Dis., 40, 588. 1927; Locke, A., and Main, E. R.: 

 ibid., 39, 484. 1926. 



