NATURE OF ANTIBODIES 203 



sensitize one unit of red cells so that they will be completely 

 hemolyzed by 0.30 c.c. of a 1 :30 dilution of guinea pis comple- 

 ment after incubation for 1 hr. in a 37° C. water bath. 



A unit of complement is the least amount of a 1:30 dilution 

 of complement that will completely hemolyze one unit of red cells 

 sensitized by two units of hemolysin. Kohner always titrates 

 complement in the presence of a test dose of antigen. 



Method of Titrating Hemolysin. — In order to determine ex- 

 l)crimentally the amount of immuiic sei-um that contains a unit of 

 hemolysin or to ascertain tlic amount of normal guinea pig serum 

 that contains one unit of complement, certain standards have to 

 be arbitrarily adoj^ted. Kolmcr, who has carried out an extensive 

 investigation of various methods used to titrate hemolysin and 

 complement, recommends that after all reagents arc added their 

 final volume shall be 3.0 c.c. in each tube. He has adopted 0.5 c.c. 

 of a 2 per cent suspension of washed slieep red cells as a iniit for 

 the red cell suspension and noriual saline containing 0.1 gm. of 

 magnesium sulphate per liter as a diluent. He makes up a series 

 of dilutions of the immune serum using a separate test tube for 

 each dilution and carefully labels each tube indicating the dilution 

 it contains. These dilutions are usually as follows: 1:1,000. 

 1:2,000. 1:3,000, 1:4,000, 1:5,000, 1:6,000, 1:8.000, 1:10,000, 

 1:12,000, 1:16,000. He then dilutes fresh normal guinea pig 

 serum (complement) 1:30 by adding one part of complement to 

 twenty-nine parts of normal saline. After this he places ten 

 Wassermann test tubes in a rack and adds the reagents accord- 

 ing to the following protocol : 



Hkmotasix Titration 



COMPLEMENT ^^^ 



TUBE HEMOLYSIX, 1:30 SOLUTION SUSPENSION 



NO. 0.5 c.c. DILUTION 



C.C. ^•^- ^•^- 



