Analyzing the Antigenic Relationship between Viruses using an Image-Based Microneutralization Assay

Add media and serially diluted receptor-destroying enzyme-treated antisera onto mammalian cell monolayers, preventing nonspecific virus binding.

Add an influenza virus suspension. Antisera antibodies, specific to viral proteins, bind, neutralizing viruses and preventing cellular entry.

Low neutralizing antibody concentration or non-neutralizing antibodies enable ineffective viral antigen binding, allowing virus attachment to cell receptors.

Discard media. Apply cellulose-containing media, forming a semi-solid overlay.

The virus uses host cell machinery to form viral particles and cause cell lysis.

The overlay restricts virus movement, facilitating localized infection and creating holes in cell monolayers.

Remove the overlay. Fix and permeabilize cells.

Wash with buffer. Add antibodies binding viral antigens within permeabilized cells.

Pipette horseradish peroxidase-conjugated secondary antibodies binding to primary antibodies.

Introduce peroxidase substrates and hydrogen peroxide, resulting in a blue product.

Image wells to quantify virus-infected cell populations against antiserum dilutions.

Identify antisera dilutions indicating a specified reduction in an infected cell population to determine neutralization titers.

To carry out virus neutralization, prepare the cell monolayer two or three days in advance, then add 50 microliters of VGM to each well of the plate. Use columns 11 and 12 for the virus control and cell control, respectively. Add 50-microliter aliquots of a 1-in-20 dilution of receptor-destroying enzyme or RDE-treated serum to the first row of columns 1 to 10.

Perform twofold serial dilutions by transferring 50 microliters from row A to row H, and discarding 50 microliters from row H. Then, add 50 microliters of diluent to each well of the cell control column. Add 50 microliters of the virus to each well of the plate except for the cell control column. Incubate the plate at 37 degrees Celsius for two to three hours. Then, remove the inoculum and add the overlay to each well. Incubate the plate overnight at 37 degrees Celsius undisturbed. Then, fix and stain the plates.

To quantify neutralization, place a well plate in the scanning area of a flatbed scanner. Scan the plate and run the well plate reader software to calculate the required viral titers.