1 82 PRACTICAL METHODS IN IMMUNITY 



antigen in Tube I. Mix thoroughly by manipulating bulb of pipette. Then trans- 

 fer 4 volumes of the i to 40 from Tube I to Tube II, and so on through the series. 

 When the dilution in the last tube has been made throw 4 volumes away. 



The 4 volumes of dilution of the antigen in the respective tubes will then be: 

 In Tube I, i to 40; in Tube II, i to 80; in III, i to 160; in IV, i to 320, and so on. 



5. Add i volume of serum to be tested to control tube X, and to each of the tubes 



I, II, III, etc., in succession. (If the serum be added to the antigen tubes before the 

 control tube, antigen might be carried over to the control.) 



NOTE. If the serum has been inactivated restore complement by adding i volume 

 of a 40% fresh guinea-pig serum. Also use 2 volumes of this inactivated human 

 serum instead of i. 



6. Incubate at 38C. for thirty minutes. This allows syphilitic antibody, if 

 present, to bind complement. 



7. As soon as the above mixtures have been made and put in the incubator 

 prepare the "haemolytic system" by adding i volume of 20% emulsion of washed 

 human red cells to 4 volumes of solution of amboceptor paper (10 Noguchi units of 

 amboceptor paper dissolved in i c.c. of salt solution. Thus of a paper of which 4 mm. 

 was the unit we should cut off about 40 mm., place in test-tube and extract the dried 

 serum with i c.c. of salt solution), and place this haemolytic system in incubator 

 alongside the tubes already there. To obtain the washed red cells allow 4 to 10 

 drops of blood to drop into a graduated centrifuge tube containing salt solution to 

 which has been added i % of sodium citrate to prevent coagulation. After shaking, 

 centrifuge. Pour off supernatant fluid, replace with salt solution, again shake and 

 centrifuge this sediment of red cells is to be diluted with 4 volumes of salt solution 

 (20% emulsion). (Incubation hastens sensitization of the red blood cells. Agglu- 

 tination of red cells may occur with certain haemolytic sera. The immunization of 

 the rabbits with small doses intravenously (Coca's method) tends to prevent this 

 interfering factor. However, frequent shaking is usually sufficient to break up the 

 agglutinating masses of red cells). 



8. At the expiration of thirty minutes from the commencement of incubation 

 for complement binding, add i volume of haemolytic system to each of the tubes, I, 



II, III, etc., in the order of antigen dilution. 



9. Finally, after washing pipette in salt solution, add i volume of haemolytic 

 system to control in tube X. (If the haemolytic system should be added to the con- 

 trol tube before the antigen tubes, complement from the control tube might be 

 carried over to the antigen tubes.) 



Shake each tube thoroughly. Allow them to incubate for a few minutes. Then 

 examine tubes I, II, III, etc., for haemolysis. The control should, of course, show 

 haemolysis. The antigen tubes should show a white, supernatant fluid over the in- 

 tact red-cell sediment in the tubes with the low dilutions and even in the highest 

 dilutions, where the serum is strongly positive. In a weakly positive serum, in- 

 hibition of haemolysis may only show in the first tube and haemolysis show in those 

 tubes having higher dilutions of antigen. 



It will be noted that the reagents are made in accordance with 

 Noguchi's directions. Even in those cases where fresh guinea-pig 

 serum is employed to replace complement, absent from the inactivated 



