528 THE TECHNIC OF COMPLEMENT-FIXATION REACTIONS 



strated by Gengou in 1902. The principles worked out by him were 

 extensively studied and practically applied by Neisser and Sachs in the 

 forensic differentiation of animal proteins. 



Hemolytic System. Complement is furnished by the fresh serum of 

 a guinea-pig diluted 1 : 20 and used in dose of 1 c.c. ( = 0.05 c.c. serum); 

 washed sheep's corpuscles are made up in a 2.5 per cent, suspension and 

 used in dose of 1 c.c.; antisheep amboceptor should be highly potent, 

 and is titrated after the method previously given (p. 399). In the 

 following titrations, and in conducting the main test the hemolytic 

 amboceptor is used in an amount equal to 2 units. 



Specific Antiserum. This is obtained from a rabbit immunized 

 with the protein for which the test is to be made, namely, human or 

 animal blood-serum. In forensic tests it may be necessary to prepare 

 a number of these antiserums with the serums of man and the ordinary 

 domestic animals. The technic of immunization is the same as that 

 employed for the preparation of precipitins (p. 70). An antiserum for 

 forensic tests must be sufficiently potent to fix complement with 0.0001 

 c.c. of its antigen. This is determined by a process of titration. If ? for 

 example, an antihuman serum is to be titrated, the method of procedure 

 is as follows : 



Secure 0.1 c.c. of fresh human serum and dilute 1 : 1000 by adding 

 99.9 c.c. of normal saline solution. Of this dilution, 0.1 c.c. is equivalent 

 to the standard dose of 0.0001 c.c. of undiluted serum. The serum may 

 be used 1 : 500. The antiserum is heated to 55 C. for half an hour and 

 diluted 1 : 10 (1 c.c. immune serum plus 9 c.c. of saline solution). De- 

 creasing doses of immune serum are mixed with a constant dose of anti- 

 gen and complement. At the same time the anticomplementary ti- 

 tration of the immune serum is made by substituting salt solution for 

 antigen. The doses to employ and the results of an actual titration 

 are shown in Table 19: 



Tube 16 is the hemolytic system control, and shows complete hemol- 

 ysis; tube 15 is the antigen control, and shows complete hemolysis, 

 as the quantity of serum is too small to exert an anticomplementary 

 influence; tubes 11 to 14 are the tests for anticomplementary action of 

 the antiserum. In the present instance the serum was several months 

 old and the maximum dose of 1 c.c. ( = 0.1 c.c. undiluted serum) was 

 very slightly anticomplementary. A fresh serum is practically never 

 anticomplementary in this dosage, but these tubes should, nevertheless, 

 * be included in each titration. Tubes 1 to 10 include the antigenic titra- 

 tion, and show that the antiserum is perfectly antigenic in dose of 



