GENERAL TECHNIC 429 



for instance, the hemolytic serum is known to have a titer of about 

 1 : 2000 (see p. 375), a stock solution is made by diluting 1 c.c. of the serum 

 with sterile salt solution to make a dilution of 1 : 200. In a series of six 

 test-tubes increasing amounts of this diluted amboceptor are placed: 

 0.05 c.c., 0.1 c.c., 0.15 c.c., 0.2 c.c., 0.3 c.c., and 0.4 c.c.; to each tube add 

 1 c.c. of complement serum diluted 1 : 20 ( = 0.05 c.c. undiluted serum) 

 and 1 c.c. of a 2.5 per cent, suspension of sheep's cells and sufficient salt 

 solution to bring the total contents up to 3 or 4 c.c. Each tube is shaken 

 gently and incubated at 37 C. for an hour. At the end of this time that 

 amount of amboceptor that just completely hemolyses the corpuscles is taken 

 as the hemolytic unit (Fig. 103). In conducting the antigen titrations 

 one and one-half or twice this amount is used as the dose. 



The antigen extract is diluted 1 : 10 by placing 1 c.c. in a test-tube and 

 slowly adding 9 c.c. of normal salt solution. 



Increasing amounts of this emulsion are placed in a series of test- 

 tubes: 0.2, 0.4, 0.6, 0.8, 1, 1.2, 1.5, and 2 c.c. To each tube are now 

 added 1 c.c. of the diluted complement serum ( = 0.05 c.c. undiluted se- 

 rum) and sufficient normal salt solution to bring the total volume up to 3 

 or 4 c.c. Shake each tube gently and incubate for one hour at 37 C. Then 

 add to each tube 1 c.c. of the corpuscle suspension and a dose of am- 

 boceptor equal to 1J/2 units, as just determined by previous titration. 

 Shake gently and reincubate for another hour and a half, when a prelim- 

 inary reading of the results may be made. That amount of antigen that 

 shows beginning inhibition of hemolysis is regarded as the anticomplemen- 

 tary unit. The final readings are made after the tubes have stood over- 

 night in a refrigerator at low temperature (Fig. 108). 



This titration may also be made in the presence of normal serum, 

 although this is not absolutely necessary. The serum must be perfectly 

 fresh, and must be that from a person known to be free from lues. It is 

 inactivated by heating to 55 C. for half an hour, and 0.1 c.c. is added 

 to each tube. Complement and salt solution are now added, and the 

 titration conducted in the manner just described. Normal serum may 

 absorb a small amount of complement in itself, and hence a titration 

 conducted with serum may show a slightly lower anticomplementary 

 dose. 



The fo/f owing controls are included: 



1. A hemolytic system control, containing the complement, corpuscles, 

 and amboceptor in the same amounts as were used in conducting the 

 titration. This control should show complete hemolysis. 



2. *A serum control, which is the same as the hemolytic system control 



