50 LABORATORY COURSE IN SERUM STUDY 



3. LIBERATION OF AMBOCEPTOR IN HAEMOLYSIS 



When red cells containing several hsemolytic doses of ambo- 

 ceptor are laked by a not excessive amount of complement, some 

 of the amboceptor is liberated in the solution. 



To 2.0 c.c. of 5 per cent sheep cells add sufficient amboceptor so 

 that there will be 20 units for every unit (0.5 c.c.) of 5 per cent sheep 

 cells. Allow to stand for one half hour. Centrifuge and wash with 

 saline three times and after the third washing make the volume again 

 up to 2.0 c.c. 



To 1.0 c.c. of cells so sensitized add 1.0 c.c. of 10 per cent guinea-pig 

 complement. Incubate one half hour. Then divide into two equal 

 portions of 1.0 c.c. each. To the first portion add 0.5 c.c. of the sen- 

 sitized cells and incubate again for one hour. Note whether haemolysis 

 of the added cells occurs. To the second portion add 0.5 c.c. of un- 

 treated corpuscle suspension and 1.0 c.c. of 10 per cent complement. 

 Haemolysis, after incubation at 37J degrees centigrade, will indicate 

 that amboceptor was liberated by the laking of the sensitized cells dur- 

 ing the first incubation and was taken up by the added cells in the second 

 incubation. 



How does the amount of amboceptor liberated compare with the 

 amount dissociated by simple incubation without haemolysis in Experi- 

 ment 1 ? 



Each student needs : 



12 c.c. of 5 % sheep cells 

 70 units of amboceptor 

 12 c.c. of complement (1-10) 

 Flask saline 



6 one-c.c. pipettes 



1 test tube rack for half-inch test tubes 



1 graduated centrifuge tube 

 20 half-inch test tubes 

 2 three-quarter-inch test tubes 



1 five-c.c. pipette 



1 nipple pipette 1 



1 If experiments 1 and 3 of this lesson are to be done on the same after- 

 noon, it is well to sensitize a considerable amount of cells at one time. 



