The cells are fixed, and virus-specific antibodies conjugated to HRP are used to visualize foci of infected cells

The cells are fixed, and virus-specific antibodies conjugated to HRP are used to visualize foci of infected cells. N.Zhu et al., 2020). In the following months, SARS-CoV-2 spread rapidly around the globe with a total of 21 million cases and 770,000 deaths (Organization, 2020). Studies defining the immune response against SARS-CoV-2 are critical for vaccine clinical trial design. Analyses of the humoral response to SARS-CoV-2 demonstrated that SARS-CoV-2 infection induces IgG and IgM antibodies directed against the receptor binding domain (RBD) of the spike protein (S) and the total spike protein (Ni et al., 2020;Okba et al., 2020;Pinto et al., 2020;Rogers et al., 2020;Suthar et al., 2020). These antibodies are capable of neutralizing SARS-CoV-2, and most patients develop neutralizing titers within 6 days of diagnosis (Suthar et al., 2020). Neutralization activity likely occurs through antibody blockade of the SARS-CoV-2 RBD with the cognate ACE-2 receptor (Ju et al., 2020;Shi et al., 2020). Pyridoxal phosphate Recent studies have shown that passive transfer of monoclonal antibodies specific for the RBD are protective against SARS-CoV-2 in mouse, hamster, and rhesus macaque challenge models (Alsoussi et al., 2020;Rogers et al., 2020;Shi et al., 2020). Thus, neutralizing antibodies are an important correlate of protection against SARS-CoV-2 infection, and a potential therapeutic for severe disease. Fast and reliable serology testing is essential for both tracking the pandemic and vaccine design. However, tools and reagents to study SARS-CoV-2 are highly limited. Many ELISA assays for measuring SARS-CoV-2 specific antibody levels have been described (Amanat et al.;Beavis et al., Pyridoxal phosphate 2020;Liu et al., 2020;Okba et al., 2020;Stadlbauer et al., 2020). However, these assays are not able to directly assess the functional capacity of SARS-CoV-2 specific antibodies. Traditional neutralization assays include plaque or cytopathic effect-based assays which require long incubation times, are technically challenging and are not amenable to rapidly screening hundreds to thousands of samples. However, recent work has demonstrated the feasibility of performing high-throughput neutralization ESM1 assays for SARS-CoV-2 (Muruato et al., 2020). Here, we describe the detailed protocol for performing a focus reduction neutralization test (FRNT) to measure the neutralization ability of SARS-CoV-2 Pyridoxal phosphate antibodies. We developed two optimized FRNT protocols for either plasma or serum, which can be performed in a 96 well plate format in 3 days. In the basic FRNT protocol, SARS-CoV-2 infected cells are visualized through an immunostaining procedure with a primary monoclonal antibody directed against the spike protein. In the alternate FRNT-mNG protocol, a mNeonGreen expressing SARS-CoV-2 virus is utilized to directly image infected cells (Xie et al., 2020). These assays provide rapid high-throughput methods to measure SARS-CoV-2 antibody neutralization. == Basic protocol 1: Focus Reduction Neutralization Test == Plaque assays are a classic methodology for detecting infectious viruses, including Betacoronaviruses. However, this method is labor-intensive, costly, low throughput, and is not efficient for performing large-scale neutralization assays on patient specimens or monoclonal antibodies. We recently developed a focus-forming assay (FFA) platform for SARS-CoV-2 (Suthar et al., 2020;Vanderheiden et al., 2020). This basic protocol provides Pyridoxal phosphate a step-by-step instruction for performing neutralization assays using this FFA platform. This protocol applies to performing a SARS-CoV-2 neutralization assay on human plasma/serum. == Materials == Vero C1008 (clone E6, ATCC, #CRL-1586) icSARS-CoV-2 (infectious cloned virus, UTMB, (Xie et al., 2020)) Horseradish peroxidase Avidin D (Vector laboratories, Inc #A-2004) Biotin labelled anti-SARS-CoV spike antibody (CR3022, Emory University, (Suthar et al., 2020)) Dulbeccos Modified Eagle Medium (VWR, #45000304) Dulbeccos Phosphate-Buffered Saline (DPBS) (VWR, #4500436) Fetal Bovine serum (Biotechne) Non-essential amino acids (Fisher Scientific, Pyridoxal phosphate #11140050) L-glutamine (VWR, 25005Cl) HEPES buffer (VWR, #4500690) Sodium pyruvate (VWR, #45000710) Antibiotics (VWR, #45000616) Trypan blue (Beta South Technologies, #T8154) Trypsin-EDTA 0.25% (Thermo Fisher Scientific, #25200072) 96 well Flat bottom cell culture treated plate (Grenier Bio-One, #655180) Falcon 96.