ANTIGEN 151 



nic. It is possibly better to start with a 1-30 dilution in the first 

 antigen tube instead of with a 1-20. It is also an advantage to titrate 

 the human complement. 



Preparation of Acetone Insoluble Antigen. Take about 50 grams of finely 

 divided beef, dog, or rabbit heart or liver and triturate in a mortar to a paste. Pour 

 on this paste 500 c.c. of absolute alcohol and keep the mixture in a corked bottle 

 in the 37 C. incubator for five to seven days. (We use beef heart and 96% alcohol.) 

 Next filter through paper and collect the filtrate in a large shallow dish and hasten 

 evaporation with the aid of a current of air from an electric fan directed upon the 

 uncovered surface. 



Within twenty-four hours only a sticky residue should remain. This is taken up 

 in about 50 c.c. of ether and the turbid ethereal solution kept over night in the refrig- 

 erator in a corked bottle. 



In the morning there will be found about 45 c.c. of clear supernatant fluid which 

 is decanted off and allowed to evaporate to about 15 c.c. 



Now to this 15 c.c. add about 150 c.c. of acetone and a precipitate will form 

 which collects at the bottom of the measuring cylinder. Now pour off the 

 supernatant acetone and let the sediment stand until it is of a resinous con- 

 sistence. Now dissolve 0.3 grams in i c.c. of ether and then add 9 c.c. of methyl 

 alcohol. This gives the stock antigen solution. 



In using the antigen solution for the Emery or Noguchi test we dilute i c.c. with 

 9 c.c. of salt solution. This opalescent, working, antigen emulsion should be made 

 up fresh on the day of preparing the tests. 



About one-half of these antigens are lacking in power to absorb 

 complement in the presence of syphilitic sera. More rarely they 

 may absorb complement with a nonsyphilitic serum (anticomplemen- 

 tary) or they may have a haemolytic action. Consequently a new stock 

 antigen should be tested as to its reliability 



1. A mixture of 0.4 c.c. working antigen emulsion, 0.6 c.c. salt solution, and o.i 

 c.c. of a 10% suspension of washed red cells when incubated at 37 C. for two hours 

 should not show any haemolysis. 



2. A mixture of 0.4 c.c. working antigen emulsion, 0.6 c.c. salt solution, o.i c.c. 

 of a 40% solution of fresh guinea-pig serum, and 2 units of amboceptor and incubated 

 at 37 C. for one hour should show haemolysis when we now add o.i c.c. of a 10% 

 washed red-cell emulsion and the whole then again incubated for two hours at 37 C. 

 (The antigen did not absorb complement in the absence of syphilitic antibodies.) 



3. A mixture of 0.2 c.c. of a i to 10 dilution of working antigen emulsion, 0.8 

 c.c. of salt solution, i drop of syphilitic serum, o.i c.c. of a 40% dilution of fresh 

 guinea-pig serum, and 2 units of amboceptor should be incubated at 37 C. for one 

 hour. When we then add o.i c.c. of a 10% suspension of washed red cells and 

 again incubate for two hours we should fail to obtain haemolysis. (The antigen can 

 absorb complement through the intermediation of syphilitic antibodies.) 



Preparation of Amboceptor Paper. In order to secure blood from the vein of a 



