Measuring the Activation of Fc-Mediated Effector Functions by HA Antibodies

To assess the ability of specific antibodies of interest to target the hemagglutinin-expressing cells, replace the supernatant of the virally infected cell cultures with 25 microliters of antibody-dependent cell-mediated cytotoxicity, or ADCC, assay medium.

Next, in a separate 96-well plate, perform 4-fold dilutions of the antibodies of interest in triplicate and fresh ADCC medium starting at 10 micrograms per milliliter according to the schematic, and taking care to include the background and no antibody controls.

When all of the antibody has been diluted, transfer 25 microliters of the diluted antibody to the appropriate corresponding wells in the experimental cell plate and place the plate in the cell culture incubator. After 15 minutes, add 7.5 times 10 to the 4th modified effector Jurkat cells, expressing the appropriate Fc receptor per well in 25 microliters of ADCC medium for a final volume of 75 microliters of ADCC medium per well.

Return the plate to the cell culture incubator for 6 hours, thawing luciferase substrate buffer in a 37-degree Celsius water bath during the last hour of the incubation. When the buffer is ready, add 75 microliters of luciferase substrate buffer to all of the wells except the background control well, and read the plate on a luminometer. The fold induction of antibody-dependent cell-mediated cytotoxicity in the transfected or infected cell cultures can then be calculated.