WASSERMANN AND OTHER COMPLEMENT FIXATION TESTS 235 



Experiment No. 2. Sensitize a rabbit to red blood cells as described in the 

 preceding chapter; obtain some of its blood, allow this blood to stand in a re- 

 frigerator until serum separates; collect the serum and put various amounts of 

 it in test-tubes from o.ooi to i.o cc., add red cells to each tube; shake the tubes 

 to mix their contents; inspect them, their appearance will be identical to that 

 observed prior to incubation in experiment No. i . 



Place the tubes in an incubator for i to 2 hours, then remove and again in- 

 spect; there will be a radical change in their appearance. Some of the tubes 

 containing the larger amounts of serum will show a clear, transparent, bright 

 red solution, the same in appearance throughout and there will be no sediment 

 whatever in these tubes. Some of the tubes containing the smallest amounts 

 of serum will present the same appearance as was observed at the end of incu- 

 bation in experiment No. i. Between these two groups of tubes will be a num- 

 ber in which, as the amount of serum increases, the amount of opaque fluid 

 diminishes, the amount of sediment diminishes and the amount of clear trans- 

 parent red fluid increases. 



When tubes to which red blood cells have been added show a clear, trans- 

 parent red fluid and no sediment, no matter how long the tubes stand at rest, 

 it is evidence that the red blood cells have been disintegrated and their hemo- 

 globin gone into solution; this change is termed hemolysis, and the tubes have 

 the same appearance as Ai, Fig. i. 



Experiment No. 2 shows that injection of red blood cells, from another 

 species, into rabbits, stimulates the production of antibodies ; that these anti- 

 bodies are present in the blood serum of such rabbits; that when this serum in 

 the fresh state and proper amount is added to red cells such as were used to 

 stimulate the production of antibodies, it will destroy or hemolize the red cells. 



Experiment No. 3. Repeat Experiment No. 2, all factors being the same 

 except that the rabbit serum be heated in a water bath at 55C. or 56C. for 

 % hour immediately before mixing with the red cells. 



There will be no hemolysis. This demonstrates that heating the serum con- 

 taining hemolytic antibodies so alters the antibodies as to deprive them of their 

 hemolytic power. 



Experiment No. 4. Repeat Experiment No. 3, all factors being the same, ex- 

 cept that in addition to placing various amounts of heated-sensitized-rabbit serum 

 and red cells in every tube, o.i cc. of fresh unheated serum, derived from an 

 untreated, unsensitized normal rabbit, is added to each tube before incubation. ' 



When these tubes are examined after incubation hemolysis will be observed 

 the same as in Experiment No. 2 evidence that the antibodies, present in the 

 serum of rabbits sensitized to red blood cells, are composed of two parts one 

 which is destroyed by heating to 5SC. for ^ hour, called complement; the other 

 which is not injured, altered or destroyed by so heating, called amboceptor. 



The experiment also shows that complement is a native constituent of all 

 rabbits, untreated and unsensitized as well as those that have been treated or 

 sensitized; and that the complement in one rabbit's serum can unite with the 

 amboceptors of another rabbit's serum. 



