Detection of Neutralizing Antibodies in Serum Samples Using a SARS‐CoV‐2 Pseudotyped Virus Assay

Conventional live virus research on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of coronavirus disease-19 (COVID-19), requires Biosafety Level 3 (BSL-3) facilities. SARS-CoV-2 pseudotyped viruses have emerged as valuable tools in virology, mimicking the entry process of the SARS-CoV-2 virus into human cells by expressing its spike glycoprotein in a surrogate system using recombinant plasmids. One significant application of this tool is in functional assays for the evaluation of neutralizing antibodies. Pseudotyped viruses have the advantage of being competent for only a single cycle of infection, providing better safety and versatility and allowing them to be studied in BSL-2 laboratories. Here, we describe three protocols for the detection of SARS-CoV-2 neutralizing antibodies through a pseudotyped virus assay. First, SARS-CoV-2 S pseudotyped viruses (PV SARS-CoV-2 S) are produced using a Moloney murine leukemia virus (MuLV) three-plasmid system. The plasmids are designed to express the GagPol packing proteins, enhanced green fluorescent protein (eGFP) as a readout system, and the SARS-CoV-2 S protein modified to remove the endoplasmic reticulum retention domain and to improve infection. Next, the internalization of PV SARS-CoV-2 S protein in human embryonic kidney 293T (HEK-293T) cells overexpressing angiotensin-converting enzyme 2 (HEK-293T-ACE2) is confirmed by fluorescence microscopy and quantified using flow cytometry. Finally, PV SARS-CoV-2 S is used to screen neutralizing antibodies in serum samples from convalescent COVID-19 patients; it can also be used for studying the cell entry mechanisms of different SARS-CoV-2 variants, evaluating antiviral agents, and designing vaccines. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Generation of PV SARS-CoV-2 S pseudotyped virus Basic Protocol 2: Assay of PV SARS-CoV-2 S internalization in target cells. Basic Protocol 3: Detection of neutralizing antibodies in serum samples.