Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

The overall goal of this enzyme-linked lectin assay is to measure functional antibody titers against neuraminidase, a glycoprotein on the influenza virus surface. This method can help answer key questions in the vaccine field, such as, Is there an antibody response to neuraminidase after influenza vaccination or infection? The main advantage of this technique is that it provides a practical platform to measure neuraminidase inhibition titers in large numbers of serum samples.

Reassortant influenza viruses that contain the neuraminidase of interest and a hemagglutinin of H6 subtype are used in ELLA. This prevents interference by antibodies against the hemagglutinin in the vaccine or the infecting infectious A virus. Working with live reassortant H6 influenza viruses requires a permit and handling under biosafety level 2 enhanced procedures.

H6 reassortant viruses that have been chemically inactivated can be used in the assay. An inactivated H6N2 virus is used in this demonstration. Prepare virus dilutions in a 96-well plate by first dispensing 120 microliters of sample diluent into columns 1 to 11 of the dilution plate.

To column 1, add an additional 96 microliters of the sample diluent. Thaw and then vortex the vial of virus before adding 24 microliters of it to a well in column 1. This gives a 1:10 dilution of virus.

Make serial two-fold dilutions of virus in the sample diluent by transferring 120 microliters from one well to the next, using clean pipette tips for each dilution. Next, wash a fetuin-coated plate three times with PBS-T. And then blot each plate onto an absorbent paper towel to remove any excess wash buffer.

Add 50 microliters of sample diluent to each well in columns 1 to 11 of the fetuin-coated plate. Add 100 microliters of sample diluent to column 12 for the negative control. Transfer 50 microliters of diluted virus from columns 1 through 11 of the dilution plate to duplicate wells of the fetuin-coated plate.

Tap gently to mix. And then cover the plate with a plate sealer. Place the plate in a humidified incubator at 37 degrees Celsius.

16 to 18 hours later, transfer the plate to the bench and remove the plate sealer. Wash the plate six times with PBS-T, then invert and pat it on absorbent paper towels to ensure that all of the liquid has been removed from the wells. Next, add 100 microliters per well of peanut agglutinin horse-radish peroxidase solution to all wells and incubate the plate for two hours at room temperature.

Less than 15 minutes before the incubation is to end, prepare the OPD solution. Wash the test plates three times to remove the PNA-HRPO and blot them dry before adding 100 microliters of the OPD substrate to each well. Incubate the plate in the dark for exactly 10 minutes at room temperature.

Stop the reaction by adding 100 microliters per well of one normal sulfuric acid. Use a plate reader to measure the optical density, or OD, at 490 nanometers for 0.1 seconds. Next, select the virus dilution that will be used for serology as described in the text protocol as well as in the results section.

It is critical to select a dilution within the linear range of the virus titration curve. We prefer a dilution that gives a signal close to maximum so that the full assay range is used. The ELLA is done after determining the dilution of virus needed for the assay.

To begin, prepare the reagents and starting materials as described in the text protocol. 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 resuspend it in the sample diluent at the dilution that was selected.

Prepare at least five 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 one in ten 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 dilutions 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.

To select the virus dilution that will be used for serology, a graph that plots the OD value at 490 nanometers at each virus dilution is generated. The maximum OD forms a plateau at the lowest virus dilutions. Select the virus dilution that gives approximately 90%of the maximum signal and is within the linear range.

Confirm that the OD at the selected dilution is at least tenfold greater than the background signal. This is the selected virus dilution for the ELLAs that employ this particular virus stock. Representative results of an enzyme-linked lectin assay showing the percent inhibition of neuraminidase activity that was calculated for each serum dilution are shown.

The serum dilution is plotted on a logarithmic scale as shown in this graph. Identify the dilution of serum that resulted in greater or equal to 50%inhibition of neuraminidase activity. In this example, the 1 in 160 dilution of serum resulted in 74%inhibition, and the 1 in 320 dilution of serum resulted in 45%inhibition.

The neuraminidase inhibition titer is therefore 160. After its development, this technique paved the way for researchers to explore antigenic differences in the neuraminidases of seasonal influenza viruses. The ELLA was also used to demonstrate that neuraminidase-inhibiting antibodies correlate with protection against clinical signs of influenza.