264 INFECTION AND IMMUNITY 



Similar alcoholic extracts of normal human spleen or of normal 

 rabbit's liver may be employed. Although often claimed that the anti- 

 gen in such extracts is furnished by the Upoids, as a matter of fact it is 

 at the present day unknown to which ingredient the immune-body 

 binding power is to be attributed. 



The antigen used in several hundred reactions by the writers with 

 satisfactory result is one prepared according to the method of 

 Noguchi/ as follows: 



Fresh normal liver or spleen is covered and thoroughly macerated 

 with five times its volume of absolute alcohol. This is allowed to extract 

 in the incubator for six to eight days, being thoroughly stirred up at 

 least once a day. It is then pressed through cheese-cloth and filtered 

 through paper. This alcoholic extract is evaporated to dryness at room 

 temperature with the aid of a wind fan. The sticky, brownish residue 

 resulting is taken up in a small quantity of ether and the solution 

 poured into four times its volume of C. P. acetone. A heavy flocculent 

 precipitate forms which settles to the bottom as a sticky brown mass. 

 This is retained as antigen and may be preserved under acetone. The 

 acetone-soluble fraction is thrown away. For use, about 0.2 gram of 

 the sticky paste is dissolved in about 5 c.c. of ether and 100 c.c. of 

 salt solution added. This is shaken until the ether has evaporated. 

 The resultant antigen, ready for use, is a slightly opalescent greenish 

 fluid from which nothing settles out on standing. 



Before an antigen can be used for the actual test, it is necessary to 

 determine the quantity which will furnish a valid result. The substances 

 which are used as antigens often have the power, if used in too large 

 quantity, of themselves binding complement. It is necessary, there- 

 fore, to determine the largest quantity of each given antigen which 

 may be used without exerting an anti-complementary action, i.e., which 

 will not inhibit in the presence of normal serum but which wiU at the 

 same time inhibit hemolysis when syphilitic serum is used. This is done 

 by mixing graded quantities of the antigen with a constant quantity 

 of complement (0.1 c.c. of fresh guinea-pig serum), in duplicate sets, 

 adding to each tube of one set 0.2 c.c. of a normal serum, and to the other 

 0.2 c.c. of a known syphilitic serum. These substances are allowed to 

 remain together for one hour and then red blood corpuscles and inac- 

 tivated hemolytic serum are added. The quantity which has given 

 complete inhibition with the syphilitic serum, but absolutely no inhibi- 



1 NogtwM, Personal communication. 



