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

1. Expression of Influenza Virus Hemagglutinin via Transfection

1. Plate Human Embryonic Kidney (HEK 293T) cells at a density of 2 x 10⁴ cells/well in a white tissue culture treated 96-well plate and let it sit for 4 h in a 37 °C incubator (with 5% CO₂).
2. Transfect cells with 100 ng of DNA coding for viral hemagglutinin and 0.2 μL of transfection reagent per well in a total volume of 50 μL.
   NOTE: Both plasmid and transfectant reagent are diluted in 1x Opti-Minimum Essential Media (MEM) Reduced Serum Medium.
3. After incubating plates in a 37 °C incubator with 5% CO₂ for 18 h, remove transfection media.

2. Assessing the FcRg/NFAT-mediated Activation of Luciferase Activity by Monoclonal Antibodies or Sera

1. First, replace the growth media of transfected cells with 25 µL of assay media (1x RPMI 1640 supplemented with 4% (vol/vol) low IgG fetal bovine sera).
2. In a separate 96-well plate, perform four-fold dilutions of the antibodies of interest starting at 30 μg/mL using the assay media. Perform dilutions in triplicates, and an example plate layout is illustrated in Figure 1A. Ensure that a control that excludes antibodies (no antibody control) and a background control (assay buffer alone) is included.
   NOTE: For both the ‘no antibody' and background controls, add 25 µL of assay buffer instead of the antibody.
3. Transfer 25 μL of the serially diluted antibodies from Step 2.2 to corresponding wells on the plate of transfected cells.
4. Incubate plate in a 37 °C incubator (with 5% CO₂) for 15 min.
5. Add 25 μL of the appropriate modified effector Jurkat cell expressing murine FcgRIV or human FcgRIIIa (75,000 cells/well). The total volume for each well should be 75 µL, and the starting final concentration of the antibody is at 10 µg/mL (Figure 1B).
   NOTE: For the background control, add 25 µL of assay buffer instead of effector cells. To examine antibody-dependent cell-mediated phagocytosis, a modified Jurkat cell expressing the human FcgRIIa can be used.
6. Incubate the plate in a 37 °C incubator with 5% CO₂ for 6 h.
7. Thaw and warm luciferase substrate buffer in 37 °C water for about 1 h before use.
   NOTE: To make the luciferase substrate buffer, reconstitute the lyophilized luciferase assay substrate in the provided assay buffer. The reconstituted substrate buffer can be stored at -30 °C to -10 °C for up to six weeks.
8. Add 75 μL/well of luciferase substrate buffer to all wells except for the background control.
9. Read on a luminometer.
10. Calculate fold induction with the following equation: Fold induction = (Induced value-Background value) / (No mAb value-Background value).