WASSERMANN AND OTHER COMPLEMENT FIXATION TESTS 237 



STANDARDIZATION OF AMBOCEPTOR 



When performing complement fixation tests one adds i cc. of a 5 per cent, 

 suspension of red blood cells to every tube, an amount of rabbit serum that con- 

 tains sufficient amboceptor to insure complete hemolysis of these cells, with 

 the amount of complement placed in the tubes, must also be added. As already 

 stated rabbits treated in an identical manner give serum which varies in ambo- 

 ceptor content of immunized rabbit serum when one procures it. To do this 

 a unit of measurement must be established and for this purpose the following 

 rule has been established: 



A unit of amboceptor is the smallest amount that will cause complete hem- 

 olysis of i cc. of a 5 per cent, suspension of red blood cells, together with i cc. 

 of a 10 per cent, solution of fresh guinea-pig serum (complement), when incu- 

 bated for i hour at 37C. 



Rabbit serum is not acceptable unless a unit of amboceptor is contained in 

 less than o.oi cc. of serum. The required accuracy of measurement cannot be 

 accomplished, under ordinary circumstances, if one attempts to discharge from 

 a graduated pipette less than o.i cc. of fluid therefore, the first step in prepar- 

 ing amboceptor for standardization is to make a i per cent, solution of the rab- 

 bit serum in normal salt solution. To 9.9 cc. of normal salt solution add o.i cc. 

 of rabbit serum, mix thoroughly. 



Take a test-tube rack built to hold 1 2 tubes and place a tube in each of the 

 first 10 holes and in the twelfth hole. 



Put o.i cc. of the i per cent, solution of rabbit serum in the first tube. 



Put 0.2 cc. of the i per cent, solution of rabbit serum in the second tube. 



Put 0.3 cc. of the i per cent, solution of rabbit serum in the third tube. 



Put 0.4 cc. of the i per cent, solution of rabbit serum in the fourth tube. 



Put 0.5 cc. of the i per cent, solution of rabbit serum in the fifth tube. 



Put 0.6 cc. of the i per cent, solution of rabbit serum in the sixth tube. 



Put 0.7 cc. of the i per cent, solution of rabbit serum in the seventh tube. 



Put 0.8 cc. of the i per cent, solution of rabbit serum in the eighth tube. 



Put 0.9 cc. of the i per cent, solution of rabbit serum in the ninth tube. 



Put i.o cc. of the i per cent, solution of rabbit serum in the tenth tube. 



Do not put any rabbit serum in the last tube. 



Put i cc. of the 10 per cent, solution of guinea-pig serum in every tube. 



Put i cc. of the 5 per cent, suspension of red blood cells in every tube. 



Shake the tubes to thoroughly mix their contents and place in incubator. 



At the end of an hour remove and inspect the tubes, the appearance may be 

 as follows: 



The first one, two or three tubes may show little or no hemolysis; the next 

 two or three tubes progressively increasing amounts of hemolysis and the 

 rest, up to and including the tenth tube, complete hemolysis, the last tube no 

 hemolysis. 



Such an observation would establish the following: Absence of anything 

 other than complement in the guinea-pig serum capable of hemolizing red cells 

 as evidenced by the last tube. 



