MODIFICATIONS OF THE WASSERMANN REACTION 479 



The antigen must be titrated as usual, and its anticomplemen- 

 tary hemolytic and antigenic properties determined. According 

 to Noguchi, an extract is satisfactory if it is antigenic in 0.02 c.c. 

 of a 1 : 10 dilution, and not anticomplementary or hemolytic in 

 amounts under 0.4 c.c. (1 : 10). In conducting the tests five times 

 the antigenic unit, or 0.1 c.c., is employed; this dose is at least four 

 times smaller than the anticomplementary unit, and is therefore safe 

 and satisfactory. 



The antigen is best preserved in methyl alcohol, as described on 

 p. 446. Dried on paper and properly preserved in sealed tubes in a cold 

 place it will retain its activity for several months, but as a general rule 

 it is best to use fresh emulsions of the alcoholic solution. 



Titration of Antigen. The anticomplementary, hemolytic, and 

 antigenic doses of an extract are determined in the same general manner 

 as was described under the Wassermann reaction. 



1. Anticomplementary Titration. A portion of the stock alcoholic 

 solution of acetone-insoluble lipoids is diluted with 9 parts of saline 

 solution. This is the emulsion that is employed in conducting the 

 Noguchi reaction, and contains 0.3 per cent, of the original lipoidal 

 substances. 



Sufficient emulsion for these titrations is prepared by diluting 0.4 

 c.c. of the alcoholic solution with 3.6 c.c. of saline solution. 



Into a series of seven small test-tubes place increasing amounts of 

 this emulsion as follows: 0.1, 0.2, 0.3, 0.4, 0.6, 0.8, 1.0 c.c., add 0.1 c.c. 

 (5 capillary drops) complement (40 per cent.) to each; also 1 c.c. of a 

 1 per cent, suspension of corpuscles and sufficient saline solution to 

 make the total volume in each tube about 2 c.c. Incubate at 37 C. 

 for one hour (one-half hour in water-bath), and add two units of ambo- 

 ceptor. Shake the tubes gently and reincubate for two hours (one hour 

 on water-bath). That tube showing beginning inhibition of hemolysis 

 contains the anticomplementary dose, which should not be under 0.4 

 c.c. In the tubes containing the larger doses slight hemolysis may be 

 noticed, which is evidence of the hemolytic action of the extract. 



An eighth tube should be included, containing 0.1 c.c. diluted com- 

 plement, two units of amboceptor, and 1 c.c. of the corpuscle suspension. 

 This is the hemolytic control and should show complete hemolysis. 



2. Antigenic Titration. Since the extract is likely to have a high 

 antigenic value, it is necessary to dilute the antigen still further by plac- 

 ing 0.5 c.c. of the foregoing emulsion in a test-tube and .adding 4.5 c.c. 

 of saline solution (1 : 100 dilution of the antigen). Into a series of six 

 test-tubes place increasing amounts of this emulsion as follows: 0.05, 



