A Microneutralization Assay to Measure Neutralizing Antibody Titers in Human Serum

For a microneutralization assay, take a multi-well plate containing heat-inactivated, serially diluted human serum with decreasing concentrations of influenza-specific neutralizing antibodies.

Introduce pathogenic influenza viruses. Serum antibodies interact with the viruses and neutralize them; however, higher serum dilutions exhibit limited virus neutralization.

Seed with Madine-Darby Canine Kidney cells, or MDCK cells, overexpressing human sialic acid residues on their surface.

In the higher dilution, non-neutralized influenza viruses fuse with the cells' sialic acid, releasing the viral genome into the cell cytoplasm.

The viral genes are transcribed and translated to viral proteins and nucleoproteins within the host cell.

Treat with a high concentration of acetone for fixation and permeabilization.

Introduce primary antibodies that interact with viral nucleoproteins.

Overlay with enzyme-conjugated secondary antibodies that interact with primary antibodies. Wash and treat with the enzyme's substrate, producing a color product.

Quantify the color intensities and plot them against the serum dilution; the highest serum dilution, achieving fifty percent virus neutralization represents the neutralization titer.

On day 1 of the MN assay, thaw the sera in a 37 degrees Celsius water bath, and remove immediately after thawing. Heat-inactivate the human sera for 30 minutes in a 56 degrees 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 to 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 of 10 diluted sera to columns A1 to A10. Perform a two-fold 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 serially 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 two-fold 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.