Antibody-dependent cytotoxicity

Mechanism: Either IgG or IgM is made against antigens. The binding of these antibodies to the surface of host cells then leads to:

1. opsonization of cells whereby phagocytes stick to host cells by way of IgG, and discharge their lysosomes and ;

2. activation of the classical complement pathway causing lysis of the cells

3. ADCC destruction of the cells whereby NK cells attach to the Fc portion of the antibodies. The NK cell then release pore-forming proteins called perforins and proteolytic enzymes called granzymes. Granzymes pass through the pores and activate the enzymes that lead to apoptosis of the infected cell by means of destruction of its structural cytoskeleton proteins and by chromosomal degradation.

Complement Fixation

The complement fixation assay can be used to look for the presence of i) specific antibody or ii) specific antigen in a patient's serum. The test utilizes sheep red blood cells (SRBC), anti-SRBC antibody and complement, along with specific antigen (if looking for antibody in serum) or specific antibody (if looking for antigen in serum). If antibody (or antigen) is present in the patient's serum, then the complement is completely utilized and SRBC lysis is minimal. However, if the antibody (or antigen) is not present in the patient's serum, then the complement binds anti-SRBC antibody and lysis of the SRBCs ensues. The following graphics illustrate the complement fixation assay.

Monoclonal Ab Production

Balb/c mice

Nuclear hybridization to give one single nucleus

Benefits of using hybridomas:

Possess the immortal growth qualities of the myeloma cell
Secrete large amounts of monoclonal Ab
Can be cultured indefinitely

How to make a M.C. Ab response to blood Cytochrome C

Take Cyto C
Inject into mouse
Take spleen B cells
Use myeloma cell to fuse with B cell (Ab)
Bring together with fusion agent PEG (polyethylene glycol)

What do you want from this?

You want to separate the hybridomas
You want Ab to Cyto C

To do this:

You have to kill off the myeloma cells (use toxic agent)
B cells will die on their own (2 weeks)

This will leave you with just the hybridomas (but a mixture of them)

You then need a selective procedure to pick out what you need.

ELISA example

Screening to see if you have B cells that are making Ab to Cyto-C
Some of the B cells will have Ab to Cyto-C, some will not.
Use 96 well plate
Plate at Conc of 100 cells/well (achieved by serial dilutions)
10 wells

The anitgen (cyto C) is coated at the bottom of each well
If there are Ab to Cyto C in the well they will attach
A secondary Ab (which is specific for the primary {the one for cyto-c}) will bind the primary essentially tagging it with an enzyme.
Addition of substrate will cause color change if the two antibody complex is present

B cells with Ab specific to Cyto-C are present in these two wells

Now we have narrowed the cells down from 1000 cells to 200 (100+100)
Over time you can take these 200 cells and through serial dilutions split them up so that you eventually have 1 B cell in every well.
Again you are using well that are coated with Cyto-C
If there is a reaction in that the well, the B cell clone must be making Ab specific to an epitope on Cyto-C