196 PRACTICAL METHODS IN IMMUNITY 



For the substratum the placenta should be obtained as soon after 

 delivery as possible or the malignant tumors as soon after operation 

 as is feasible. All connective tissue, blood, etc., should be removed 

 from the tissue from which the substratum is to be prepared. 



The tissue is then cut up finely, put in a deep jar and washed several times with 

 distilled water. It is then boiled in a great excess of distilled water 100 times as 

 much and boiling continued about thirty minutes. This water is decanted and 

 the tissue added to fresh boiling water, repeating the process five or six times until 

 when placed in a dialyzing thimble no ninhydrin reacting soluble proteins can be 

 recognized in the dialysate by the blue color. 



The sterile tissue is now placed in a jar, covered with distilled water and on top 

 of this toluol is deposited to maintain the sterility. To estimate the proper amount 

 of substratum to use in a test one must determine this using normal as well as 

 specific sera, recognizing the quantitative error of the test. The amount usually 

 called for is 0.5 gram. 



Bronfenbrenner is of the opinion and has supported it by much 

 experimental work that it is not digestion of the substratum which 

 takes place but autodigestion of the specific serum when placed in 

 contact with its substratum, the products of such autodigestion being 

 of the nature of anaphylatoxins. 



Anaphylatoxin Production Test. Bronf enbrenner's Modification of 

 Abderhalden's Test. Bronfenbrenner found that if he treated the 

 serum of an animal with a particular substratum and then injected 

 the autodigested serum intradermically he obtained a skin reaction. 

 This reaction, however, only obtained for homologous animals, those 

 of a different species not reacting satisfactorily. 



He now reports satisfactory results with the following technic: About 2 c.c. of 

 the patient's serum is injected intraperitoneally into a guinea-pig, thus passively 

 transferring to the guinea-pig the specific substances of the human patient's serum. 

 The next day the guinea-pig is bled, its serum collected and placed on ice with a 

 suitable amount of substratum (placenta, bazillen emulsion, tumor tissue, etc.). 



Eighteen hours later the serum is separated from the substratum by centrifugaliza- 

 tion, pipetted off and placed in the incubator for fifteen hours. At this time inject 

 0.05 c.c. of the autodigested serum, possibly containing anaphylatoxin, into the 

 shaven skin of a normal guinea-pig. 



From twelve to twenty-four hours later a distinct skin reaction occurs at the site 

 of injection, provided the serum of the patient contained antibodies. 



Instead of the skin reaction one may inject 0.5 c.c. of such autodigested serum 

 into the heart or vein of a normal guinea-pig and death will result of anaphylactic 

 shock. 



If the patient's serum were negative the injection of as much as 5 c.c. would not 

 cause symptoms. 



