Epitope Titering

The previous procedures (see Basic Protocols 1 and 2 and Alternate Protocols 1 and 2) determine the antibody titer at a constant epitope concentration. The working titer of any antibody is also dependent upon the concentration of epitopes. The optimal antibody concentration required to saturate cells having 105 epitopes is different from that required to saturate cells having 104 epitopes, and both cell types can be present in the same suspension. To determine this titer, the antibody concentration is held constant and the epitope concentration is varied by increasing the cell concentration.

1. Adjust target cell concentration to 128 x 109/ml and prepare four serial 1/4 dilutions to produce 64 (neat), 16, 4, and 1 x 106 cells in 50 |l of PBS.

If blood or bone marrow leukocytes are used as target cells, add 5 ml of cell solution to a 50-ml centrifuge tube containing 45 ml ammonium chloride lysing solution (appendix2A). Centrifuge 5 min at 1500 x g, 4°C. Remove the supernatant and resuspend cells in residual solution, adjusting the concentration of the suspension before making serial dilutions.

2. Mix 50 |l of cell suspension with 10 |l of antibody whose titer was determined using 0.5-1 x 106 cells. Incubate 15 min at 4°C.

3. Add 3 ml PBS. Centrifuge tubes 3 min at 1500 x g, 4°C.

4. Remove supernatant and resuspend cells in residual solution.

5. Add the amount of 2% ultrapure formaldehyde in PBS needed to give a final concentration of 106 cells/ml.

6. Acquire 2000 target cells using a flow cytometer.

7. Display histogram for each dilution.

8. Adjust markers using auto sample (or isotype control sample) so that <1% of events are above the marker.

9. Determine the mean channel linear fluorescence intensity (MCF) of both positive (signal) and negative (noise) cells for the four tubes.

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