An Immunofluorescence Method to Measure the Neutralization of Human Cytomegalovirus
Transcrição
Take serial dilutions of antibodies specific for human cytomegalovirus, or HCMV.
Upon adding HCMV, the antibodies bind to glycoprotein B on the viral envelope, neutralizing the virus.
Transfer the mixture onto human fibroblasts and incubate.
A trimer glycoprotein complex on the non-neutralized viruses binds to specific cellular receptors, triggering glycoprotein B-mediated virus-host membrane fusion and releasing the genomic DNA-containing nucleocapsid inside.
The released genome enters the nucleus, encoding nuclear-localized immediately early, or IE, proteins.
Antibody-neutralization prevents viral entry into the cells.
Remove the non-internalized viruses. Treat with ethanol at a sub-zero temperature to fix and permeabilize the cells.
Introduce a primary antibody that binds to the IE proteins.
Add a fluorophore-conjugated secondary antibody that binds to the primary antibody and a fluorescent stain that binds to DNA.
Under a microscope, detect dual-stained cells that are virus-infected. Calculate the percentage of infected cells to determine virus neutralization at the different antibody concentrations.
