178 PRACTICAL METHODS IN IMMUNITY 



The standardization of the titer is similar to that for the human hsemolytic 

 amboceptor, except that 5% emulsion of sheep cells with 0.05 c.c. undiluted guinea- 

 pig serum is used instead of the %% emulsion of human cells and the o.i c.c. of 40% 

 guinea-pig serum. 



The method is to take in a test-tube 0.2 c.c. inactivated human 

 serum (heated for twenty minutes at 56C.), o.i c.c. fresh serum from 

 guinea-pig for complement, i unit antigen and 3 c.c. normal salt solu- 

 tion; then to incubate for one hour at 37C. (An antigen unit is the 

 amount that will inhibit haemolysis of i c.c. of 5% emulsion of sheep 

 cells when mixed with 0.2 c.c. luetic serum and o.i c.c. guinea-pig 

 complement.) 



There should likewise be absolutely no inhibition with 0.02 c.c. of serum from a 

 normal individual. The unit is made up to i c.c. with salt solution. 



Then add 2 units of amboceptor and i c.c. 5% emulsion of sheep 

 red cells, shake and incubate for one hour. (The amount of haemolytic 

 serum that will haemolize i c.c. of a 5% emulsion of sheep red cells 

 to which 0.05 c.c. guinea-pig serum has been added, in one hour, is an 

 amboceptor unit.) 



In our laboratory we use i c.c. of a i to 10 dilution of the clear stock acetone 

 insoluble antigen of Noguchi as the antigen unit. 



In the preparation of the antigen originally recommended by Wasser- 

 mann, we finely divide the liver of a syphilitic foetus and extract it for 

 twenty-four hours with salt solution containing 0.5% carbolic acid. 

 Frequent shaking is required. The supernatant fluid is decanted and 

 centrifuged. This turbid fluid is pipetted off and kept in the ice-box in 

 a brown bottle for several days. This yellowish-brown opalescent fluid 

 is the antigen and is standardized so that i unit equals the amount 

 which will inhibit haemolysis of i c.c. of 5% emulsion of sheep red 

 cells when mixed with 0.2 c.c. luetic serum, o.i c.c. guinea-pig com- 

 plement and 2 units of amboceptor. 



The same technic is employed with the control test-tube except that 

 the antigen unit is not put in. 



The Noguchi method has been stated to give a positive reaction with nonsyphilitic 

 sera in about 7% of cases. The Wassermann a negative result in about 9% of syph- 

 ilitic sera. These figures show the advantage of checking one against the other. 



One objection to the Wassermann test is that a majority of human 

 sera show native antisheep amboceptors and in some instances the 

 amount of this constituent may be so great, when added to the 2 units 



