160 ANTIGENS AND THE TECH NIC OF SERUM REACTIONS 
(c) The visible hemolysis that results from the activation of an 
erythrocyte— inactivated specific immune serum complex by non- 
specific complement. 
The general plan of procedure is to incubate an antigen (as bac- 
terial cells) and inactivated serum and complement in proper pro- 
portions for one hour, to permit the three components to unite. A 
mixture of erythrocytes and specific inactivated hemolytic serum is 
now added. If the reactive substances are properly proportioned 
and the inactivated serum first added is specific for the antigen (bac- 
teria), no hemolysis will occur when the hemolytic system is added, 
because all the complement present is bound by the bacteria-immune 
serum complex. On the contrary, if the inactivated serum is not 
specific for the bacterial antigen, no union between the two will take 
place, complement will not be bound, and it is free in the mixture. It 
will activate the erythrocyte-inactivated immune serum complex, and 
hemolysis will occur. 
It will be seen that the hemolytic system is added as an indicator. 
The absence of hemolysis shows a union of bacterial antigen, inactive 
specific bacterial immune serum and complement. Hemolysis shows 
that the union has not taken place, the complement, therefore, was free 
in the mixture, and it united with the hemolytic system, causing 
hemolysis in the erythrocyte antigen through the specific amboceptor or 
hemolysin. 
The method of complement-fixation may be employed to examine 
sera for specific antibodies, using a known antigen, or to test sus- 
pected antigens with sera containing specific known antibodies. The 
most practical application of the method in medicine is in the serum 
diagnosis of syphilis, glanders and other bacterial infections. 
The Technique of Complement-fixation.^ The technique of comple- 
ment-fixation is simple in principle, but it requires the most scrupu- 
lous attention to details. All glassware must be neutral in reaction, 
chemically clean, and bacteriologically sterile. Physiological salt 
solution (0.85 to 0.9 per cent C. P. XaCl in neutral, freshly distilled 
water) used for washing red blood cells and for dilutions should be 
sterile and stored in clean containers. It should be freshly prepared; 
upon standing CO2 is gradually absorbed, which tends to interfere with 
the reaction. 
The Wassermann Serum Diagnosis of Syphilis.— Five elements enter 
into the Wassermann test for syphilis: the antigen, suspected syphilitic 
serum, complement and a hemolytic system consisting of red blood 
cells and specific inactivated immune hemolytic serum (hemolysin). 
Preparation and Standardization of A ntici en. ^The antigen originally 
employed by Wassermann and his collaborators was an aqueous 
extract of syphilitic tissue which was prepared by suspending 1 part by 
weight of finely comminuted liver of a syphilitic fetus^ in 5 parts of 
1 The tissue is examined for the specific organism; if Treponemata are abundant it 
is converted into antigen, otherwise it is discarded. 
