17() ANTIGENS AND THE TECH NIC OF SERUM REACTIONS 
hearts differ in their content of antigenic hpoids. The titration of an 
antigen with salt sohition tends to overcome differences in Hpoid con- 
tent. Thus, an antigen rich in hpoids will require more dilution with 
saline to bring it to the required titer than an antigen weak in lipoids. 
Titration alone does not always bring an antigen to the required degree 
of sensiti^'eness, hence the necessity for correction. 
When an antigen is more sensitive than the standard, the antigen is 
diluted with alcohol containing 0.6 per cent cholesterol. The amount 
of cholesterolized alcohol to be employed is determined by trial. Thus, 
to a given amount of the antigen is added perhaps 15 per cent chol- 
esterolized alcohol and the final product is tested with sera, employing 
standard antigen as a control. If still more sensitive, further alcohol 
dilution is tried until the sensitiveness of the antigen is reduced to that 
of the standard. 
An antigen may be less sensitive than the standard because of either 
excessive or deficient concentration of lipoids. Those cases where the 
undersensitiveness is due to insufficient lipoids usually show a titer 
requiring less than 1 cc. of saline per cubic centimeter of antigen. 
Antigens of this type require the addition of ether extractives to 
increase the concentration of lipoids. To this end, the ether extracts 
of 25 gm. beef heart are evaporated with the aid of a fan to about 25 cc. 
and filtered. To a small amount of the unknown antigen is added 
0.5 per cent of the concentrated ether extract and the product tested 
with serum, using standard antigen as a control. As indicated by the 
results obtained, more or less concentration with the ether extractives 
is to be employed. If an antigen is less sensitive because of excessive 
concentration of lipoids— and such an antigen usually shows a titer 
higher than 1 cc. saline per cubic centimeter of antigen— its sensitive- 
ness is increased to that of standard antigen by dilution with alcohol. 
An illustration of the correction of this type of an antigen is given in 
the table, page 177. 
It should be stated that it is not necessary to retitrate an antigen 
after correction. The use of an arbitrary titer of 1 cc. antigen + 1.1 cc. 
saline will be found to give good results. 
It is suggested that the standardization of antigen should be carried 
out in central laboratories. It requires the same effort to standardize 
50 cc. as 50 liters, and once standardized, an antigen will keep at least 
five years and very likely indefinitely. Antigen should be kept tightly 
stoppered in the dark at room temperature and the stoppers should be 
covered with thin, high-grade tin foil. Extreme cold may cause the 
separation of cholesterol or other lipoids, which may, however, be 
redissolved by warming the container in a water-bath or incubator. 
Serum.— The blood specimen should be centrifuged to remove clot 
and cells. The serum must be entirely free from cells or other particles. 
Previous to its use in the test, the serum is heated in a water-bath at 
56° C. for thirty minutes. When serinn that has been heated is kept 
over night in the ice-box, reheating for ten minutes at 56° C. is neces- 
sary before using in the test, 
