Encyclopedia of Experiments: Immunology
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Transcript
Take tubes with a recombinant hepatitis C virus, or HCV suspension containing RNA encoding a secreted luciferase reporter.
Add an antiviral test compound and solvent to the test and negative control tubes.
The test compounds bind to HCV glycoproteins, inactivating the virus.
Post-incubation, dilute the mixtures to prevent compound-cell interactions in subsequent steps.
Add these diluted mixtures onto HCV-replication-supporting hepatoma monolayers. Incubate.
The compound-inactivated HCV cannot attach to specific cell surface receptors, while active HCV glycoproteins bind to these receptors, facilitating cellular attachment.
Remove unadsorbed HCVs. Wash with buffer and add media. Incubate for a prolonged duration.
The bound HCV is internalized, releasing viral RNA and leading to viral protein and luciferase synthesis, secreted from the cell.
Collect the luciferase-containing culture supernatants. Centrifuge and add a luciferase substrate to the supernatants.
Luciferase oxidizes the substrate, emitting light.
A higher luminescence in the control well than in the test indicates viral inactivation by the compound.
