ACTION OF ANTITOXINS ON TOXINS. 269 



intervene. As sensitized corpuscles are employed the anti-alexic 

 serum should be without effect on the sensitizer used for these 

 corpuscles. The heated normal serum used as control to the anti- 

 alexin or the sensitizer should have no particular properties and 

 should be simply an inert fluid. And of course the absence of hemo- 

 lytic activity in the heated sera should be controlled. The reagent 

 employed (sensitized red blood corpuscles) should be added only 

 in very small doses in order that the smallest amounts of free alexin 

 may be detected. 



To fulfill these conditions we use the following materials: As 

 alexin, fresh guinea-pig serum is employed. As anti-alexin, the 

 heated serum of a rabbit that has been given injections two or 

 three times of fresh normal guinea-pig serum. For sensitized cor- 

 puscles hen red cells washed in salt solution and treated with rabbit 

 > hen serum, 56 degrees, are employed. As controls of the anti- 

 alexin and the sensitizer, normal rabbit serum, 55 to 56 degrees. 



In short, the toxin (alexin) is from the guinea-pig. The other 

 sera (antitoxin, sensitizer and control) are all from the rabbit and 

 have no effect on one another. No accessory reactions, therefore, 

 should destroy the accuracy of the experiment. 



The strength of the anti-alexin is first defined by the following 

 experiment : 



Tube a. Alexin, 0.2 of a cubic centimeter; anti-alexin (55 degrees), 0.3 of a 

 cubic centimeter. 



Tube b. Alexin, 0.2 of a cubic centimeter; normal rabbit serum (55 degrees), 

 0.3 of a cubic centimeter. 



An hour or two later 0.2 of a cubic centimeter of sensitizer (serum 

 rabbit > hen, 56 degrees) is added to each tube and then one drop 

 of washed hen blood. The corpuscles are hemolyzed in 20 minutes 

 in "6." There is no hemolysis in "a," even on the following day. 



This lack of hemolysis in u a" may be shown to be due solely to 

 a neutralization of the alexin. The sensitizer, present in relatively 

 large amount in respect to the corpuscles, is unaffected.* 



Tube a. Sensitizer, 0.1 c.c; anti-alexin, 2 c.c; fresh rabbit serum (alexin), 2 c.c. 

 Tube b. Same as "a," with 2 c.c. normal rabbit serum, 55 degrees, replacing 

 the anti-alexin. 



* This is shown by replacing the guinea-pig alexin, which is specifically affected 

 by the anti-alexin, by another alexin, say from the rabbit, on which the anti-alexin 

 has no effect. 



