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.