Encyclopedia of Experiments: Immunology
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Transcript
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.
