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

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 …

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