162 



LABORATORY COURSE IN SERUM STUDY 



A. TlTRATION OF TRYPSIN SOLUTION 



In each of a series of test tubes place decreasing amounts of trypsin 

 solution, 1 c.c., 0.7, 0.5, 0.4, 0.3 and 0.2 c.c. Add sufficient salt solution 

 to bring up the volumes to 2.5 c.c. and set up a control containing 2.5 c.c. 

 of saline alone. To each tube add 2 c.c. of casein solution, shake, and 

 place in water bath for half an hour at 37 C. Remove the tubes from 

 the water bath and add 3 to 4 drops of the acetic acid solution to each. 

 The tube with the smallest amount of trypsin which shows no precipi- 

 tate of undigested casern contains the amount of trypsin needed for 

 the subsequent test. 



PRELIMINARY TITRATION OF TRYPSIN 



B. DETERMINATION OF ANTITRYPTIC ACTIVITY OF SERUM 



For control a mixture of several normal sera is used and a 2 per cent 

 solution in normal saline is prepared. A 2 per cent solution of the serum 

 to be tested, preferably from a case of advanced carcinoma, is used for 

 the test. 



In order to demonstrate the lipoidal nature of normal antitrypsin it 

 can be removed from serum by chloroform extraction. 4 c.c. of diluted 

 normal serum should be placed in a test tube, 0.5 c.c. of chloroform added, 

 the mixture thoroughly shaken and incubated for one hour, at the end 

 of which time the mixture is centrifugalized and the clear supernatant 

 fluid pipetted off. The normal serum, the carcinoma serum, and the 

 normal serum after chloroform extraction should be tested in parallel 

 as follows : 



In each of six tubes place 0.5 c.c. of -the 2 per cent solution of serum. 

 To the first tube add the dose of trypsin which will completely digest the 

 casein as determined by previous titration (this is "the unit" of trypsin), 

 and in the successive tubes increasing amounts of the trypsin so that 

 Tube 6 contains four times as much trypsin as Tube 1. For example, if 



