Luciferase-Based Pseudovirus Neutralization Assay

Resumen

Source: Chang, X. et al. Preparation of Pseudo-Typed H5 Avian Influenza Viruses with Calcium Phosphate Transfection Method and Measurement of Antibody Neutralizing Activity. J. Vis. Exp. (2021)

The video demonstrates the neutralization of influenza pseudoviruses by antibodies that block the HA protein from binding to host cells. A luciferase reporter system reveals the reduction in viral entry with increasing antibody concentrations, showing the effectiveness of antibody-mediated neutralization.

Protocolo

Pseudovirus neutralization (PN) assay

1. Make single-cell suspensions of MDCK cells (5 x 10⁴ per mL) in complete Dulbecco's Modified Eagle Medium (DMEM) medium, and add 100 µL of the cell suspension to each well of a 96-well flat-bottom plate. Incubate the cells in a 37 °C, 5% CO₂ incubator for 20 h.
2. Take out the pseudovirus and sera from the -80 °C refrigerator, thaw it on the ice, and vortex thoroughly.
3. Dilute the sera in the complete medium 20 times, and add 100 µL of complete DMEM medium to the wells of a 96-well round-bottom plate from columns 2-10.
4. Add 200 µL of the diluted sera into the wells of column 2, transfer 100 µL from column 2 to column 3, dilute the sera as 1:20, 1:40, 1:80, etc., to 1:10240 successively from column 2 to column 10, and discard the 100 µL from column 10.
5. Add 100 µL of DMEM complete medium to the wells of column 11 as the negative control.
6. Add 100 µL of pseudoviruses to each well, pipette up and down the mixture 5 times thoroughly, and incubate the mixture plate in a 37 °C, 5% CO₂ for 1 h.
7. Transfer 100 µL of the mixture from the 96-well round-bottom plate to the corresponding well of the 96-well cell culture plate. Put the plate back in the incubator (37 °C, 5% CO₂) for 60 h.
8. Take the plate out of the incubator. Perform the luciferase assay.

Materiales

Lista de materiales utilizados en este artículo

Nombre	Empresa	Número de catálogo	Comentarios
96-well cell culture plates (flat-bottom)	Thermo fisher scientific	167008	consumable material
96-well cell culture plates (round-bottom)	Thermo fisher scientific	163320	consumable material
Dulbecco’s Modified Eagle Medium (DMEM)	Gibco	12100-046	Reagent
Fetal Bovine Serum	Gibco	16000-044	Reagent
Luciferase Assay System Freezer Pack	Promega	E4530	Reagent kit
MDCK.1	ATCC	CRL-2935	Cell line
Penicillin-Streptomycin	Gibco	15140-122	Reagent
Pipette Tips (10 μL)	Thermo fisher scientific	TF102-10-Q	consumable material
Pipette Tips (100 μL)	Thermo fisher scientific	TF113-100-Q	consumable material
Pipette Tips (1000 μL)	Thermo fisher scientific	TF112-1000-Q	consumable material
Serological pipets (5 mL)	Thermo fisher scientific	170355N	consumable material
Serological pipets (10 mL)	Thermo fisher scientific	170356N	consumable material
Trypsin/EDTA	Gibco	25200-072	Reagent