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

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.