An Assay to Measure Influenza Neuraminidase Inhibition Antibody Titers

Take multi-well plate wells coated with a glycoprotein that acts as a substrate for neuraminidase or NA, surface proteins of the influenza virus.

Add serially diluted heat-inactivated human serum containing NA-inhibiting antibodies to test wells and media alone to the control well.

Introduce the virus and incubate.

In the control well, viral NA cleaves the glycoprotein's terminal sialic acid, exposing the penultimate galactose. In the test wells, antibody binding inhibits NA activity.

Remove the virus-serum mix. Add lectin-peroxidase conjugates, which bind to the exposed galactose.

Remove the unbound conjugates and add a chromogenic peroxidase substrate mixed with hydrogen peroxide.

Immobilized peroxidase utilizes hydrogen peroxide to oxidize the substrate into a colored product.

Add acid to lower the pH, stopping the reaction.

Measure the optical density and calculate the percent NA inhibition.

The reciprocal of the serum dilution needed for fifty percent NA inhibition is the NA-inhibiting antibody titer.

Heat and activate the serum samples in a water bath at 56 degrees Celsius for 45 to 60 minutes. Thaw a vial of virus, vortex, and resuspended in the sample diluent at the dilutio