An Assay to Measure the Neutralizing Antibody Titer Specific for Respiratory Syncytial Virus

Take serially diluted, heat-inactivated human serum containing respiratory syncytial virus or RSV-specific neutralizing antibodies.

Add the viral suspension. The antibodies neutralize the viruses in a dilution-dependent manner.

Introduce the mixture to human epithelial cell monolayers and incubate for viral adsorption.

Discard the mixture. Add carboxymethylcellulose-containing media, forming a semi-solid overlay. Incubate.

RSV utilizes the host cell machinery to synthesize viral RNA and proteins, which subsequently assemble and bud as individual virions.

Infected cells eventually die. Carboxymethylcellulose restricts virus movement, causing localized infection and cell death, forming plaques.

Remove the overlay.

Fix the cells with an acetone-containing buffer. Introduce a detergent-containing blocking buffer to permeabilize the cells and prevent non-specific antibody interactions.

Add primary antibodies targeting viral proteins.

Overlay with fluorophore-conjugated secondary antibodies targeting the primary antibodies.

Image the wells to quantify fluorescent plaques.

The highest serum dilution neutralizing fifty percent of RSV represents the neutralization titer.

Prior to the inoculation of A549 cells with RSV, examine the cell plate under a light microscope to confirm that A549 cell monolayers are 80% confluent. Then, invert the plate gently to discard the media, and lightly blot the plate on a sterile, absorbent paper towel. After each wash with filtered, sterile PBS.

Next, add 100 microliters per well of the virus-serum mixture to the corresponding wells on the A549 cell plate according to the plate template provided in the manuscript, and incubate the plate at 37 degrees Celsius in 5% carbon dioxide for one hour. At the end of the incubation, use a pipette to remove 90 microliters per well, and add 100 microliters of the media containing 1X M 199, 1.5% CMC, and 2% FCS and penicillin-streptomycin mixture. Incubate the plate at 37 degrees Celsius in 5% carbon dioxide for three days.

To fix and develop an assay plate, first invert the RSV-infected A549 cell plate gently to discard the media containing M 199, CMC, 2% FCS, and penicillin-streptomycin mixture. Then, slowly add 200 microliters of the fixation buffer to each well. Incubate the plate at minus 20 degrees Celsius for 20 minutes. Then, discard the fixation buffer, and gently blot on an absorbent paper towel.

Leave the plate face down to dry for 10 minutes. Then, with filtered PBS buffer containing 0.05 polysorbate-20, make 5% milk-blocking solution. Add 200 microliters of the blocking solution to each well, and incubate the plate at room temperature for 30 minutes. At the end of incubation, invert the plate gently to discard the solution, and blot on an absorbent paper towel.

To make the primary antibody, dilute 1 to 500 Goat X RSV antibody in the blocking solution. Add 50 microliters to each well, and incubate the plate at 37 degrees Celsius for one hour. Wash the plate three times with filtered PBS polysorbate.

Next, make the secondary antibody by diluting 1 to 5,000 AlexaFluor Donkey anti-Goat immunoglobulin G in the blocking solution. Add 50 microliters to each well, and incubate at 37 degrees Celsius for one hour. Wash the plate five times with filtered PBS polysorbate.

To count plaques with an automated spots reader, first, use an unused 96-well culture plate to calibrate the instrument by entering the count settings in the FITC channel. Then, read the cell plate, wrap it in foil, and store it at 4 degrees Celsius. Next, check the images of wells for artifactual plaques or disrupted cell monolayers, and exclude wells with disrupted cell monolayers.