174 ANTIGENS AND THE TECH NIC OF SERUM REACTIONS 
has been dissolved, the antigen is filtered. The cholesterolized extract 
is stable and may be stored in the dark indefinitely at room temperature. 
The ether and alcohol emplo}'ed in antigen preparation should be of 
high purity and the latter should be at least 95 per cent. Corks 
covered with thin, high-grade tinfoil have been found satisfactory as 
stoppers for flasks used in antigen preparation and storing. Rubber 
as well as cork stoppers give off soluble elements into the antigen which 
modify the final product. 
Standardization of Antigen.— The aim of standardization is to bring an 
antigen to the same degree of sensitiveness as that possessed by standard 
antigen. This degree of sensitiveness was established as a result of 
clinical and serological studies. It is essential to establish a standard 
because of variations in content of antigenic lipoids in heart muscle. 
Antigen standardization involves the following steps: (1) Titration of 
antigen. This determines the smallest amount of normal saline which, 
when added to 1 cc. of antigen will produce a suspension of lipoid 
particles capable of solution (dispersion) in additional salt solution. 
(2) Determination of antigen sensitiveness. This is carried out by 
testing the antigen at its titer with different sera, employing standard 
antigen as a control. If the sensitiveness is greater or less than the 
standard, correction is resorted to. (3) Correction of antigen. This 
involves decreasing or increasing the lipoid concentration of an antigen 
until the standard degree of sensitiveness is obtained. 
Titration of Antigen.— One cubic centimeter of cholesterolized antigen 
is added to each of five standard antigen suspension vials. To five 
similar vials are added in order the following amounts of physiological 
salt solution: 0.8, 0.9, 1, 1.1 and 1.2 cc. Each vial of salt solution is 
emptied into a given antigen vial and without waiting to drain the salt 
solution, the mixture is immediately poured back and forth five or six 
times to permit of thorough mixing. Each of the five antigen suspen- 
sions will show the presence of definite particles. 
The antigen suspensions are allowed to stand for thirty minutes after 
which the solubility in salt solution of the lipoid particles is tested as 
follows: 0.05, 0.025 and 0.0125 cc. amounts, respectively, of each 
antigen suspension are pipetted with a 0.2 cc. pipette graduated in 
0.001 cc. into the bottom of each of three standard tubes. Into each 
tube is then measured 0.15 cc. quantities of physiological salt solution. 
The rack is shaken vigorously for three minutes after which salt solu- 
tion is added to each tube— 1 cc. to the 0.05 cc. antigen suspension 
amount and 0.5 cc, to the others— and observation made as to whether 
or not the original antigen-suspension particles have become dissolved. 
Of the tubes containing an opalescent solution free from visible par- 
ticles (redispersion of the suspension), that 3-tube group is selected as 
the end-point or titer in which the proportion of saline to antigen is a 
minimum. The accompanying table gives an outline of a typical 
antigen titration. 
