378 HEMOLYSINS 



Methods for Removing Hemolysins from a Serum. In general, 

 these aim to remove the natural hemolysins, such as natural antisheep 

 hemolysin, from human serums preliminary to making the Wassermann 

 test, or from a guinea-pig serum that is to foe used as complement. 



The method of removal consists simply in adding corpuscles to the 

 serum, and allowing sufficient time for the corresponding hemolytic 

 amboceptor to become attached and then removing both by centrifuging 

 the mixture. If the serum is fresh, it should be cooled to to 3 C., 

 in order to inhibit complement activity, which would hemolyze a por- 

 tion of the corpuscles. 



To remove natural antisheep hemolysin from a patient's serum place 

 a measured quantity of cold serum in a centrifuge tube and add four 

 \ volumes of a 2.5 per cent, suspension of sheep's cells. This will make a 

 dilution of 1 : 5, so that 0.5 c.c. of the dilution is equivalent to 0.1 c.c. of 

 undiluted serum. After placing the tube in ice-water for from fifteen 

 to thirty minutes centrifuge thoroughly to remove the corpuscles. The 

 process may be carried out after the serum has been inactivated, in 

 which case it is not necessary to work with cold serum. 



In removing a natural hemolytic amboceptor from a guinea-pig 

 serum that is to be used as complement a measured amount of serum is 

 first removed to a separate tube and thoroughly chilled in a glass of 

 cracked ice. If a large amount of serum is to be used, for example, 5 c.c., 

 it is well to place about 0.1 to 0.2 c.c. of pure undiluted corpuscles, after 

 their last washing, in the bottom of a centrifuge tube. This quantity 

 of corpuscles does not materially affect the dilution of the serum. If a 

 smaller amount of serum is used, such as 1 c.c., it is well to add 8 c.c. of a 

 2.5 per cent, suspension of corpuscles, and after centrifuging the mixture 

 the final dilution of 1 : 20 is secured by adding 10 c.c. of salt solution to 

 the supernatant diluted serum. 



Method of Determining Natural Hemolysins in Serum. To ascer- 

 tain whether or not a certain natural amboceptor is present in a serum it 

 is merely necessary to inactivate the serum, and to a measured amount, 

 for example, 0.2 c.c., add 1 c.c. of complement serum (1 : 20) that is 

 known to be free of the particular amboceptor in question, and 1 c.c. 

 of a 2.5 per cent, suspension of the corresponding corpuscles. Sufficient 

 salt solution is added to bring the total volume to 3 or 4 c.c. The mix- 

 ture is then incubated at 37 C. for one or two hours, when the occur- 

 rence of hemolysis indicates the presence of the amboceptor for the 

 corpuscles employed. 



To determine the amount of natural amboceptor by titration dilute 



