530 THE TECHNIC OF COMPLEMENT-FIXATION REACTIONS 



The Test. Into a series of six small test-tubes place increasing doses 

 of extract of the blood-stain (antigen), as follows: 0.1 c.c., 0.2 c.c., 0.4 

 c.c., 0.6 c.c., 0.8 c.c., 1 c.c.; add double the titrated dose of antiserum 

 and 1 c.c. of complement (1 : 20), with sufficient salt solution to bring 

 the total volume in each tube up to 3 c.c. 



The following controls are included : 



1. Antigen control; 1.0 c.c. of the blood extract plus 1 c.c. of diluted 

 complement and salt solution. 



2. Antiserum control: double the titrated dose plus 1 c.c. of diluted 

 complement and salt solution. 



3. Hemolytic control; at this time, 1 c.c. of diluted complement and 

 salt solution. 



4. Corpuscle control: 1 c.c. of corpuscle suspension and salt solution. 

 The tube should be plugged with cotton. 



Shake all the tubes gently and incubate for an hour at 37 C. in a 

 water-bath or, preferably, six hours or overnight in a refrigerator at 

 8 C. Add 2 units of hemolytic amboceptor and 1 c.c. of corpuscle 

 suspension to each tube except the corpuscle control. Shake gently 

 and reincubate for from one to two hours in a water-bath, depending 

 upon the degree of hemolysis present in the controls. 



The readings are made at once, and again after the tubes have been 

 allowed to settle in the refrigerator overnight. Inhibition of hemolysis 

 with the smallest dose of blood extract 0.1 c.c. ( = approximately 0.0001 

 c.c. of blood) indicates that the blood extract is most certainly the 

 antigen for the antiserum employed. Even with the maximum dose of 

 extract 1 c.c. ( = approximately 0.001 c.c. of blood) inhibition of 

 hemolysis serves to show the nature of the blood. With an antihuman 

 serum, for instance, a similar specific reaction would be possible only 

 with the bloods of the higher apes. 



In making blood tests for medicolegal purposes the antiserum 

 should not only be standardized with a definite dilution of human serum, 

 but the whole test should first be conducted with a known dried human 

 blood-stain, and it must be borne in mind that extreme accuracy in all 

 manipulations is essential. 



In Table 20 are shown the method and the results of an actual test, 

 using a dried human blood-stain and the same antiserum as previously 

 directed. 



I prefer this complement-fixation test to the precipitin reaction in 

 the differentiation of proteins, as the readings are sharper and more 

 definite. This test is fully as reliable as the precipitin test, and there 

 is less danger of group reaction. 



