162 ANTIGENS AND THE TECHNIC OF SERUM REACTIONS 
lecithins and related substances. It keeps well and appears to be very 
sensitive and reliable. From 0.2 to 0.3 gm. are dissolved in a mixture 
of 1 cc. of ether (free from alcohol and having a neutral reaction) and 10 
cc. of neutral absolute methyl alcohol. This solution is kept in an 
amber bottle in the refrigerator as a stock antigen. One cubic centi- 
meter of this stock antigen is added to 19 cc. of physiological salt 
solution; this is the antigen used for the test. 
Before making a test it is necessary to standardize the antigen. 
It is essential to know the anticomplementary titer, that is, the maxi- 
mum amount of antigen which will inhibit hemolysis in the presence 
of syphilitic serum, but which will not inhibit hemolysis when non- 
syphilitic serum is used. In addition, the following determinations 
are sometimes desirable. 
The hemolytic titer, that amount of antigen which will of itself 
cause lysis of the red blood cells, and the antigenic titer, the amount 
of complement it will absorb or "fix" in the presence of a definite 
amount of specific syphilitic serum. 
The anticomplementary titration is made by mixing graded amounts 
of antigen and a constant amount of complement (0.1 cc. of 10 per 
cent solution^ with constant amounts (0.1 cc.) of known syphilitic 
serum and normal serum, both inactivated. 
The various mixtures are incubated in a water-bath at 37° C. for 
one hour, then 1 cc. of red blood cell suspension and 1.5 units of inac- 
tivated hemolytic serum are added and again incubated in the water- 
bath at 37° C. The maximum amount of antigen which will give 
complete inhibition of hemolysis with syphilitic serum and no inhibi- 
tion of hemolysis in the non-syphilitic serum is regarded as the unit. 
Example of an Anticomplementary Titration op Antigen. 
Normal 
Comple- 
Hemolytic 
serum 
ment, 10 
Red blood 
serum in- 
Tube. 
Antigen. 
inactive, 
cc. 
per cent, 
cc. 
cells, cc. 
activated 
units. 
Result. 
1 
0.2 
0.1 
0.1 
1.0 
1.5 
1 
Complete hemolysis. 
2 
0.4 
0.1 
0.1 
d'i 
1.0 
1.5 
O 
" " 
3 
4 
0.6 
0.8 
0.1 
0.1 
0.1 
0.1 
hi 
1.0 
1.0 
1.5 
1.5 
o 
fo 3 
a 
5 
1.0 
0.1 
0.1 
JS -^ 
1.0 
1.5 
j3 ^ 
Partial inhibition. 
6 
1.5 
0.1 
0.1 
J2 g 
1.0 
1.5 
Marked inhibition. 
7 
2.0 
0.1 
0.1 
ll 
1.0 
1.5 
i o 
Complete inhibition. 
82 
0.1 
0.1 
1.0 
1.5 
03 
Complete hemolysis. 
9' 
0.1 
^§ 
1.0 
1.5 
^ 
Thus, in the experiment cited. Tube 5, containing 1 cc. antigen, 
shows beginning inhibition of hemolysis. This is regarded as the 
anticomplementary titer of the antigen. 
1 Prepared by adding fresh normal guinea-pig serum to physiological salt solution in 
the proportion of 1 part serum to 9 parts salt. 
2 Serum control. ^ Hemolytic control. 
