ANTITRYPSIN TEST 251 



salt solution up to 500 c.c. Filter and sterilize in an Arnold sterilizer. 

 Preserve in the refrigerator. 



Acetic Acid Solution. To 5 c.c. of acetic acid (c. p.), add 45 c.c. 

 of absolute alcohol and 50 c.c. of distilled water. 



The patient's serum must be fresh, and should be diluted 20 times 

 with salt solution. Dose, 0.2 c.c. 



Technic. A titration of the trypsin solution must precede the test 

 proper. Into each of several small test-tubes place increasing amounts 

 of trypsin solution, as, for example, 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 c.c. 

 Add 2 c.c. of the casein solution to each tube; shake carefully and place 

 in an incubator or water-bath for half an hour at 50 C. Then add 

 three or four drops of the acetic acid solution to each tube, and observe 

 which tube first shows cloudiness after a few minutes. The tube con- 

 taining the smallest amount of trypsin and which remains perfectly 

 clear contains enough trypsin fully to digest the 2 c.c. of casein solution. 



Into each of six small test-tubes now place 0.2 c.c. of the 1 : 20 dilu- 

 tion of the patient's serum, and increasing amounts of the trypsin solu- 

 tion, beginning with the completely digesting dose, as determined above, 

 and increasing by 0.1 c.c. Add 2 c.c. of casein solution to each tube, 

 and bring all tubes to a like volume by the addition of normal salt solu- 

 tion. Shake gently, and incubate at 50 C. for half an hour. Add 

 several drops of acetic acid solution to each tube, and again observe the 

 tube containing the smallest amount of trypsin in which cloudiness can 

 be seen. In this way the amount of trypsin neutralized by the anti- 

 trypsin of the serum is determined. 



For example, in an experiment the preliminary titration showed that 

 0.5 c.c. of trypsin completely digested the casein. In the second part 

 of the test the lower limit of trypsin was this 0.5 c.c. increased by 0.1 

 c.c. in successive tubes up to 1 c.c. It is now found that 1 c.c. of the 

 trypsin solution is required to bring about the complete digestion of the 

 casein in the presence of the serum, or 1 c.c. 0.5 c.c. =0.5 c.c., which is 

 the amount of trypsin neutralized by 0.01 c.c. of undiluted serum. 



A control experiment is conducted with the pooled serum of several 

 normal persons, and a comparison of the value thus obtained shows 

 whether the antitryptic power of the serum tested is altered. 



The method of Marcus, 1 which is a modification of the method of 

 Muller and Jochmann, 2 is described in the laboratory exercises on 

 Experimental Infection and Immunity. 



1 Berl. klin. Wochschr., 1908, No. 4; 1909. 



2 Munch, med. Wochschr., 1909, Nos. 29 and 31. 



