AMBOCEPTORS AND COMPLEMENTS 925 



jcctions each day; four days after the last injection the rabbit blood is titrated and 

 usually contains a fair amount of anticat hemolysin. 



2. Heat 2 c.c. of the immune serum at 56 C. for thirty minutes and inject into 

 the external jugular vein of a cat. 



3. Place the animal in a metabolic cage and collect the urine. 



4. After two or three days, autopsy, removing the spleen, liver, and kidneys. 

 Place in 5 per cent, formalin and prepare and stain sections with hematoxylin and 

 eosin and Giemsa solution. 



(a) Has blood destruction occurred? What are the evidences? 



(b) Would hemolysis occur in the test-tube with a heated immune 

 serum? If not, why not? 



(c) How then do you explain hemolysis in vivo with a heated serum? 



(d) Examine sections of the spleen, liver, and kidneys. Are there 

 any evidences of phagocytosis of blood-cells, focal necrosis, and nephritis? 

 Explain the probable mechanism of the production of these changes. 



EXERCISE 28. AMBOCEPTORS AND COMPLEMENTS. HEMOLYSINS 

 EXPERIMENT 73. TITRATION OF A HEMOLYTIC AMBOCEPTOR 



1. Secure 1 or 2 c.c. of blood from the ear of a rabbit which has been immunized 

 with injections of sheep cells. Separate v the serum and inactivate by heating to 56 

 C. for half an hour. Dilute 1: 100 by adding 0.1 c.c. serum to 9.9 c.c. normal salt 

 solution. 



2. Prepare 40 c.c. of a 5 per cent, dilution oi fresh guinea-pig serum to be used for 

 complement. Prepare 40 c.c. of a 23^ per cent, suspension of washed sheep cells by 

 adding 1 c.c. of corpuscles to 39 c.c. of normal salt solution. 



3. Proceed with the titration as given in the text on page 397. If the smallest 

 dose, viz., 0.1 c.c. of the 1 : 100 dilution, completely hemolyzes the corpuscles, it will be 

 necessary to retitrate with a dilution of 1 : 1000. 



(a) Is it necessary to use exactly the same amounts of complement 

 and corpuscles in all tubes and if so, why? 



(b) If hemolysis did not occur at all in this experiment, what fac- 

 tors may be at fault? 



(c) If the corpuscle control were completely hemolyzed, what de- 

 duction would you draw? 



(d) What is the amboceptor unit of this serum? 



EXPERIMENT 74. QUANTITATIVE FACTORS IN SERUM HEMOLYSIS 



1. Having determined the amboceptor unit of the above antisheep immune serum, 

 proceed as follows: 



2. To a series of four test-tubes add an amount of immune serum equaling one, 

 two, three, and five amboceptor units respectively. To each tube add 0.5 c.c. of the 

 1 : 20 dilution of complement serum. This amount is just half the dose of comple- 

 ment used in titrating the amboceptor. Add 1 c.c. of a 2J^ per cent, suspension of 

 sheep cells to each tube and sufficient salt solution. 



