Assessing Functional SARS-CoV-2 Neutralizing Antibodies

Begin with serially diluted human serum containing varying concentrations of neutralizing antibodies against severe acute respiratory syndrome coronavirus 2 or SARS-CoV-2.

Introduce pseudotyped viruses with luciferase RNA, integrase, and reverse transcriptase, encapsulated within a lipid bilayer containing SARS-CoV-2 spike proteins.

Antibodies neutralize the viruses based on serum dilution, with lower dilutions exhibiting maximum neutralization.

Introduce target cells expressing angiotensin-converting enzyme-2 receptors or ACE2 receptors and transmembrane protease serine-2 or TMPRSS2.

In higher serum dilutions, non-neutralized viruses interact with ACE2 receptors, while TMPRSS2 cleaves the spike protein, enabling virus fusion.

Upon entry, luciferase RNA is reverse-transcribed to DNA that integrates into the host genome and produces luciferase enzyme.

Replace the medium with an assay buffer containing luciferase substrate and a lysing agent.

The cells lyse, releasing intracellular luciferase, which interacts with the substrate and produces high luminescence.

Quantify luminescence across serum dilutions to determine viral neutralization percentage — the dilution achieving fifty percent neutralization represents the neutralization titer.