This study outlines a method to assess neuraminidase (NA) inhibition using heat-inactivated human serum. The assay measures the optical density of a colored product formed by the reaction of lectin-peroxidase conjugates with exposed galactose after NA activity is inhibited by antibodies.
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 dilution that was selected. Prepare at least 5 milliliters of virus for each assay plate.
Keep the diluted virus on ice, until the plates are washed, and serum samples have been added to the plate. Prepare dilutions of the serum samples and any controls, by making serial two-fold dilutions in a round-bottom 96-well plate, beginning with a 1-in-10 10 dilution in sample diluent.
Use a multichannel pipette to transfer 50 microliters of each serum control or sample dilution from the dilution plate into duplicate wells of a washed fetuin-coated plate. The sample dilution should be added in columns 2 to 11. Add 50 microliters of diluted virus to all wells, except for the negative control in column 12.
Add 50 microliters of sample diluent to wells in column 1, and add 100 microliters of sample diluent to column 12. Cover the wells with a plate sealer, and then mix by gently tapping the sides of the plate or by placing it on a plate shaker at moderate speed for 10 seconds.
Place the plate in a humidified incubator at 37 degrees Celsius for 16 to 18 hours. When the overnight incubation is complete, wash the plate before adding PNA-HRPO, and complete the assay as before. Read the optical density at 490 nanometers, and confirm that the assay results are valid. Determine the 50% endpoint titer, as described in the text protocol and the results section.
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