250 THE PRINCIPLES OF IMMUNOLOGY 



preserved under toluol. The dialyzing thimbles are especially prepared for 

 work of this kind. They are kept in distilled water for at least a week and 

 are carefully tested before use so as to be sure that protein does not pass 

 through and also to be sure that pepton will pass through. For the actual 

 test a dialyzing thimble is placed in a clean, dry, sterile Erlenmeyer flask. 

 About 0.5 gram of dried substratum is placed in the bottom of the thimble 

 and 1.5 c.c. of serum then introduced. The thimble is withdrawn, closed at 

 the top by means of a forceps and the outside washed carefully with sterile 

 water so as to remove any adherent protein. The thimble is then replaced in 

 a flask containing about 20 c.c. sterile distilled water. The contents of the 

 thimble and the water in the flask are covered with toluol and the flask 

 incubated for sixteen to eighteen hours. The dialyzate is examined by 

 means of the ninhydrin test. For this purpose, 0.2 c.c. of i per cent, ninhydrin 

 solution is placed in a clean dry test tube and 10.0 c.c. of the dialyzate added, 

 the mixture boiled for one minute and the color observed. The development 

 of a blue or violet color indicates the presence of diffusible protein products 

 and constitutes a positive test. Proper controls of all the reagents are essential. 



Since the earlier work of Abderhalden appeared, numerous articles 

 have been written and there has been much discussion concerning the 

 alleged specificity of the reaction. The protective ferments of Abderhal- 

 den are assumed to possess the property of directly digesting the antigen 

 and the appearance of the products of such direct digestion constitutes 

 the fundamental principle of the Abderhalden test. Stephan, Haupt- 

 mann, Bronfenbrenner and others have shown that these ferments lose 

 their activity after heating to 58 C. for one-half hour, but they can 

 be reactivated by the addition of fresh serum. This suggests a parallel 

 with the activity of complement and amboceptor, but Frank and Rosen- 

 thai point out that in hemolysis there is no indication that the action 

 of complement is accompanied by proteolysis. Therefore, although the 

 ferment may be reactivated after heating, this does not necessarily 

 indicate that it is of the nature of an amboceptor or other immune 

 body. Flatow, Plaut and others have reported that positive results 

 can be obtained by the manipulation of material and that positive or 

 negative reactions can thus be found with almost any serum. De Waele 

 found that he could demonstrate a digesting substance within a few 

 minutes after the parenteral introduction of foreign protein, a time 

 interval too short for the production of specific ferments. Heilner and 

 Petri regard this, however, as a sort of mobilization of ferment and not 

 the result of new formation. Bronfenbrenner found that the serum of 

 highly immunized animals failed to digest the protein used for im- 

 munization. He determined, however, that such a serum gave a positive 

 Abderhalden test and with his collaborators has demonstrated that the 

 dialyzable substances do not originate from the substratum. He showed 

 also that the ferments responsible for the cleavage of protein during 

 the reaction are not specific. Positive results with placenta are to be 

 obtained with the serum of males as well as of females, but the protein 

 digested is that of the serum. The work of Jobling and his collabor- 

 ators favors the view that proteolytic activity of the serum is not 

 specific. Plaut, Bronfenbrenner and others found that positive 

 Abderhalden tests may be obtained by the use of kaolin, starch, barium 

 sulphate and chloroform, all of which probably absorb the inhibiting 

 substance or antiferment of the blood. Van Slyke and his associates, 



