564 THE RELATION OF COLLOIDS AND LIPOIDS TO IMMUNITY 



The exact nature of the reaction is not known. The antigens are 

 soluble in alcohol, but their nature is obscure. The antibody involved 

 in the reaction is referred to as the miostagmin, but its relation to other 

 antibodies is also unknown. It is probably a physicochemical or col- 

 loidal reaction, and for this reason it has been placed in this chapter. 



Technic. The antigen is most difficult to prepare. A recent method 

 described by Ascoli is as follows: 



1. Cut non-degenerated portions of malignant tumor (cancer or 

 sarcoma) into small pieces and dry in vacuo or spread out in a thin 

 layer on clean glass plates and keep at a temperature of 37 C. 



2. Pulverize the dried substance and extract with pure methyl 

 alcohol (in the proportion of 5 gm. to 25 c.c.) for twenty-four hours at 

 50 C. in closed vessels, and shake occasionally. 



3. Filter while still hot, and allow the filtrate to cool, and then filter 

 again through Schleicher and Schull's filter-paper No. 590. 



4. It is now necessary to titrate the antigen and to determine in what 

 dilution it should be employed. Various dilutions of the antigen are 

 made with distilled water, as, e. g. } 1 : 10, 1 : 25, 1 : 50, 1 : 100, 1 : 150, 

 1 : 200, etc. A fresh normal serum is diluted 1 : 20 with normal salt 

 solution, and 9 c.c. of this are mixed with 1 c.c. of the various antigen 

 dilution. Into another tube place 9 c.c. of the diluted serum and 1 c.c. 

 of distilled water. All test-tubes, pipets, and other glassware used must 

 be perfectly dry. 



The tubes are gently shaken and placed in an incubator at 37 C. 

 for two hours. The drop number for each fluid is then estimated by 

 Traube's stalagmometer. This instrument is merely a finely and 

 elaborately graduated pipet with a central bulbous reservoir. The 

 dropping end of the instrument ends in a flattened ground base, thus 

 insuring uniformity in the size of the drops. The instrument is so 

 graduated that a fraction of a drop can be estimated. That antigen is 

 to be chosen that does not alter the drop number for normal serum by more 

 than one drop in a cubic centimeter the strongest dilution that fulfils this 

 condition being chosen. 



The Test. The patient's serum is diluted 1 : 20 with normal salt 

 solution and its drop number determined. Then take two tubes, and 

 into one place 9 c.c. of diluted serum plus 1 c.c. of antigen dilution; 

 into the other place 9 c.c. of diluted serum plus 1 c.c. of distilled water. 

 A third tube may be prepared, which should contain 9 c.c. of normal 

 serum (1 : 20) plus 1 c.c. of the same antigen dilution. A fourth tube 

 contains 9 c.c. of a known positive serum (1 : 20) from a case of cancer 

 and 1 c.c. of the antigen dilution. 



