The McGill Physiology Virtual Lab

Immunology Laboratory

Complement mediated cytotoxicity
 

Complement mediated cytotoxicity is an effector function assay. Antibody by itself is rather ineffectual in eliminating foreign organisms. However, antibody of the IgM and IgG class can activate the Complement (C') system resulting in a stimulation of different effector functions such as phagocytosis and lysis of foreign organisms.

  If red blood cells are used as a source of antigen it is relatively simple to demonstrate the lytic properties of the antigen-antibody-complement complex.
Procedure
 Take 5 tubes and label them 1 to 5. In each tube place 0.3 ml of SRBC (1% solution). In tubes 2 and 3 place 0.3 ml of antiserum and in tube 4 place 0.3 ml of normal mouse serum. In tube 1 place 0.6 ml of saline and tubes 2 and 5 place 0.3 ml of saline. In tubes 3, 4 and 5 add 0.3 ml of complement solution. Place in the water bath at 37C and incubate for 30 minutes. Remove from the water bath and centrifuge. Record the colour of the solution in each tube and explain the reason for the differences observed.

Tube:

1

2

3

4

5

Add:

0.3 ml SRBC (1%)

 

0.3ml anti-serum

0.3ml anti-serum

0.3ml normal mouse serum

 

0.6ml saline

0.3ml saline

 

 

0.3ml saline

 

 

 

0.3ml Cí

0.3ml Cí

0.3ml Cí

Incubate:

In water bath for 30 minutes

Centrifuge:

 

Record:

Colour change

After recording your results, take tubes 2 and 4 and add 3 ml of saline to each tube and gently resuspend the RBC pellet with a Pasteur pipette. Centrifuge them gently for 5 minutes to form a RBC pellet. Remove the tubes from the centrifuge and aspirate off the supernatant with a Pasteur pipette.
To each tube add 0.3 ml of saline, and to tube 2 add 0.3 ml of C' solution and to tube 4 add 0.3 ml of antiserum. Mix gently with a Pasteur pipette and incubate at 37C (water bath) for 30 minutes, centrifuge for 5 minutes. Record the colour change and interpret your results.
What do you conclude about the specificity of antibody and complement binding?

Tube:

2

4

Add:

0.3 ml saline (after washing the cells, centrifuging, removing the supernatant)

 0.3ml C'

 

0.3ml antiserum

 

Mix gently with Pasteur pipette

Incubate:

In water bath for 30 minutes

Centrifuge:

 5 minutes

Record:

Colour change

Results
Upon centrifugation, the cells "made up" in clear saline will collect into a pellet at the bottom of the tube and since no lysis has occurred, there will be a clear saline supernatant over the pellet (left tube).

If lysis has occurred, the release of sheep red blood cell contents in the supernatant will occur and a "ghost" pellet made up of cell membrane debris will be at the bottom of the tube (right). Which sequence of events caused cell lysis?

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