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 …

Divulgazioni

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

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Citazione di questo articolo
Measuring the Activation of Fc-Mediated Effector Functions by HA Antibodies. J. Vis. Exp. (Pending Publication), e22083, doi: (2024).

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