Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

The overall goal of this microneutralization assay is to measure neutralizing antibody responses to contemporary H3N2 Influenza viruses in human sera using MDCK-SIAT1 cells. This method can be used to answer key questions to evaluate influenza immunity. For example, it can determine whether influenza vaccination induces sufficient neutralizing antibody responses to circulating H3N2 viruses, to prevent infection.

The main advantage of this technique is that it utilizes MDCK-SIAT1 cells and microneutralization assays to detect antibody responses to reset antigenic clusters of H3N2 influenza viruses. Virus stocks should be propagated to high infectious titers prior to use in microneutralization assays. Begin this procedure with propagation of AH3N2 viruses in MDCK-SIAT1 cells, as detailed in the text protocol.

On day one, thaw a vial of virus at room temperature, and immediately place the virus on ice. To test the virus at two different starting dilutions, first add 100 microliters of virus to 99 milliliters of virus diluent for the 10:2 pre-dilution. Then add 1 milliliter of the 10:2 pre-dilution to 9 milliliters of virus diluent for the 10:3 pre-dilution.

Using two microtiter plates, add 100 microliters of micro-diluent to all wells, except column one of the 96 well microtiter plate. Next, perform half-log 10 dilutions. Add 146 microliters of the virus starting dilution to all wells in column one, and serially transfer 46 microliters from column one through column eleven.

Change pipette tips between columns. After mixing column eleven, discard the tips with the 46 microliter dilution. Use column twelve as the cell control which only contains virus diluent.

Then incubate the plate for one hour, at 37 degrees celsius, 5%carbon dioxide. Prepare the MDCK-SIAT1 cells as described in the text protocol. Add 100 microliters of the diluted cells to each well of the microtiter plates.

And cover the plates. Incubate the cells at 37 degrees celsius, 5%carbon dioxide, for 18 to 20 hours. On day two, perform the ELISA, by first removing the medium from the microtiter plates.

After washing each well with 200 microliters of PBS, add 300 microliters of cold 80%acetone to each well. And incubate at room temperature for 10 minutes. Then, remove the fixative.

And allow the plates to air dry. Next, dilute the anti-influenza A nucleoprotein monoclonal antibody to the target concentration in the antibody diluent. Wash the plates three times with 300 microliters of wash buffer.

Then add 100 microliters of diluted primary antibody to each well. Incubate the plates at room temperature for one hour. For the secondary antibody addition, dilute the goat anti-mouse IgG conjugated to HRP antibody to the target concentration in the antibody diluent.

Wash the plates three times with 300 microliters of wash buffer. Add 100 microliter of diluted secondary antibody to each well, before incubating the plate at room temperature for one hour. Next, wash the plates five times with 300 microliters of wash buffer.

And tap on a lint-free wipe. Add 100 microliters of freshly prepared substrate to each well. And incubate at room temperature until the color development saturates and the optical density or OD of the cell control wells is less than 0.2.

Then add 100 microliters of stop solution to all wells. Read the OD of the wells at 490 nanometers using a microplate spectrophotometer. Calculate the TCID50 of the virus using the Reed-Muench method as described in the text protocol.

On day one of the MN assay, thaw the sera in a 37 degree celsius water bath and remove immediately after thawing. Heat and activate the human sera for 30 minutes in a 56 degree celsius water bath, as described in the text protocol. Then place the sera on ice and add the virus diluent to the sera to achieve a 1:10 pre-dilution.

To test with one virus, add 50 microliters of virus diluent to rows B through H, except columns 11 and 12. Then, add 100 microliters of 1:10 diluted sera to columns A1 to A10. Perform a twofold serial dilution from rows A through H.And discard the last 50 microliters at row H.For the virus control, add 50 microliters of virus diluent to wells A12, B12, C12 and D12.

For the cell control, add 100 microliters of virus diluent to wells E12, F12, G12 and H12. For the control sera, add 100 microliters of diluted control sera to well A column 11. And add 50 microliters of virus diluent to wells B11 through H11.

Proceed to serial dilute down. Cover the plates and incubate at 37 degrees celsius, 5%carbon dioxide, until ready for the virus addition. For virus addition, dilute the virus to 100 TCID50 in 50 microliters with virus diluent.

Add 50 microliters of diluted virus to all wells, except for column 11 on the back titration plates and the cell control wells, E12, F12, G12, and H12 on all plates. Add 50 microliters of virus diluent to all wells in column 11. Then add 50 microliters of the virus at 100 TCID50 in 50 microliters to the first well.

Next, mix and transfer 50 microliters to successive wells to perform twofold serial dilutions. Change the pipette tips between wells to avoid virus carryover. Discard 50 microliters from well H11.

Then, add 50 microliters of virus diluent to column 11 to bring the final volume to 100 microliters. Tap the plates to mix. Incubate the plates at 37 degrees celsius, 5%carbon dioxide, for one hour, before performing MDCK cell addition on day one and ELISA on day two, as before.

After adding the primary and secondary antibodies, as described in the text protocol, perform substrate addition and plate reading. Wash the plates five times with 300 microliters of wash buffer and tap on a lint-free wipe. Add 100 microliters of freshly prepared OPD substrate to each well.

Then, incubate the plates at room temperature until the virus control wells reach an optical density at 490 nanometers between 0.8 to 3 with the cell control at a low background OD less than 0.2. Next, add 100 microliters of the stop solution to all wells. Read the OD of the wells at 490 nanometers using a microplate spectrophotometer.

Finally, determine the neutralizing antibody titer of each serum sample, as described in the text protocol. Representative results of the microneutralization assays are shown here. The OD in each well represents the amount of virus infection and replication in MDCK-SIAT1 cells, in the presence of serially diluted sera containing neutralizing antibodies.

The 50%neutralization cutoff is defined as the median OD of virus control wells plus the median OD of cell control wells, divided by two, the reciprocal of the highest serum dilution that achieves equal or greater than 50%neutralization is considered the antibody titer for the serum sample. In this example, paired sera collected pre and post influenza vaccination from two patients, were tested against H3N2 virus used in vaccine, during the 2016 to 2017 influenza season. With patient 1 prevaccination sera, none of the sera dilutions inhibited virus infection and so it is considered negative.

Following vaccination, sera from this patient demonstrated neutralization. The third dilution from this sera is the highest dilution below the 50%cutoff, therefore this patient has a postvaccination titer of 40. In comparison, serum from the second patient prevaccination contains preexisting neutralizing antibodies at a titer of 320.

Following vaccination, the titer increased to greater than 1280. In this case, at a 1:10 pre-dilution of serum, no antibody end titer was achieved. The serum can be retested at a higher pre-dilution in order to achieve end titer.

Microneutralization assays using MDCK-SIAT1 cells are highly sensitive, specific and robust in detecting neutralizing antibody responses for recent antigenic clusters of H3N2 influenza viruses. By using regular MDCK cells, this procedure can also be used to detect antibody responses to other strains of H3N2 viruses and other subtypes of influenza viruses, to assess protective immunity to influenza.