Measuring the Activation of Fc-Mediated Effector Functions by HA Antibodies
Published: March 29, 2024
Abstract
Source: Bailey, M. J., et al. A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies. J. Vis. Exp. (2018).
This video demonstrates an assay measuring the antibody-mediated activation of T cell effector functions. Adding an influenza virus hemagglutinin (HA)-specific monoclonal antibody to transfected mammalian cells expressing HA results in the formation of immune complexes with HA. Upon introducing engineered T cells expressing Fc receptors and a luciferase reporter, the T cells are activated by the antibody-HA complexes, and this activation is detected by adding a luciferase substrate and measuring the produced bioluminescence.
Protocol
1. Expression of Influenza Virus Hemagglutinin via Transfection 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). 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 …
Representative Results
Figure 1: A schematic of a sample plate layout and composition of the experimental groups. (A) The starting concentration for each antibody sample is at 10 µg/mL and serially diluted across the plate (column 1 to column 12) 4-fold. Each sample is done in triplicates. Row G is reserved for the no mAb control, and Row H is the background. (B) The final volume …
Disclosures
The authors have nothing to disclose.
Materials
| HEK 293T cells | ATCC | CRL-3216 | Human embryonic kidney cells |
| Lipofectamine 2000 | Life Technologies | 11668019 | Transfection reagent |
| 1X Opti-MEM Reduced Serum Medium | ThermoFisher Scientific | 51985-034 | |
| White tissue culture treated 96-well plate | Corning, Inc. | 3917 | Assay plates |
| 10X MEM | Gibco | 11430-030 | |
| Jurkat cell expressing murine FcgRIV | Promega | M1201 | Kit provides includes cells, Bio-Glo Luciferase assay system, 1X RPMI and low IgG serum |
| Jurkat cell expressing human FcgRIIIa | Promega | G7010 | Kit provides includes cells, Bio-Glo Luciferase assay system, 1X RPMI and low IgG serum |
| Jurkat cell expressing human FcgRIIa | Promega | G9901 | Kit provides includes cells, Bio-Glo Luciferase assay system, 1X RPMI and low IgG serum |
| Luminometer | BioTek | Synergy H1 Multi-Mode reader | Luminescence plate reader |
| Bio-Glo Luciferase Assay System | Promega | G7940 | Contains luciferase assay buffer and luciferase assay substrate |
Tags
Cite This Article
Measuring the Activation of Fc-Mediated Effector Functions by HA Antibodies. J. Vis. Exp. (Pending Publication), e22083, doi: (2024).