This study investigates the neutralization of human cytomegalovirus (HCMV) using specific antibodies. The methodology includes serial dilutions of antibodies and subsequent infection of human fibroblasts to assess viral entry and infection rates.
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.
On the day before the experiment, prepare target cells for infection by culturing fibroblast cells, grown in complete media, consisting of DMEM supplemented with 10% FBS, in a sterile 96-well plate.
After 24 hours, prepare the virus-antibody mixture by adding 90 microliters of diluted antibody solution into the wells of a fresh 96-well plate. Prepare serial dilutions of antibody solution using complete media.
Additionally, add 60 microliters of the virus solution, prediluted in complete media, into each antibody-containing well to generate various concentrations of the virus-antibody mixtures and incubate.
Now, to infect the target cells, take the plate seeded with fibroblasts. Add 100 microliters of different concentrations of the virus-antibody mixture to the cells, and incubate the target plate for 24 hours.
After 24 hours, to detect the viral infection using immunofluorescence, fix the cells with absolute ethanol and incubate them in -20 degrees Celsius freezer. Post-fixation, proceed with the subsequent steps on the laboratory bench.
Pipette 100 µl of the primary antibody to all the wells of the plate. Place the plate at room temperature for an hour.
Remove the primary antibody solution from the wells. Add a mixture of fluorophore-conjugated secondary antibody and DAPI solution to all the wells. Incubate the plate for an hour in the dark, and wash the plate.
To quantify infection, visualize the plate under a microscope, and acquire images using the blue and red channels. Calculate the percentage of infection per well, as detailed in the text protocol.
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