Luciferase-Based Pseudovirus Neutralization Assay

Take a suspension of a non-replicating pseudovirus that displays the influenza hemagglutinin (HA) glycoprotein used for host cell entry and carries a luciferase reporter gene.

Add equal amounts of the viral suspension to the serially diluted immune serum containing HA-specific antibodies, and co-incubate.

These antibodies bind to the HA protein and block its receptor-binding sites, preventing viral attachment and neutralizing the pseudovirus.

High antibody concentrations neutralize most pseudoviruses, while lower antibody concentrations leave more pseudoviruses unneutralized.

Add the pseudovirus-serum mixtures to a multi-well plate containing mammalian epithelial cell monolayers and incubate.

The unneutralized pseudoviruses attach to the cells and enter them, expressing the reporter gene to produce the luciferase enzyme.

The neutralized pseudoviruses fail to enter the host cells.

Higher neutralization results in lower luciferase expression, whereas lower neutralization results in higher luciferase expression.

The samples are now ready for the luciferase assay to quantify pseudovirus neutralization.

Add 100 microliters of the single cell suspension of MDCK cells to each well of a flat bottom