SARS-CoV-2 Neutralization Assay with IgGs

Neutralization assays with serum IgGs against the 12-strain global virus panel, were performed in 96-well plates following published protocols^{9 (link),90 (link)}. To this end, 12 HIV-1 pseudovirus strains were each mixed with 1:2 serial dilutions of purified IgG (1 mg/ml starting concentration, 8 dilutions) and incubated for 1 h at 37 °C. TZM-bl cells (RRID:CVCL_B478; ordered from the HIV Reagent Program, Cat-No. ARP-8129) were added (10⁴ per well) in growth medium (DMEM, Gibco; 10% heat-inactivated FBS, Sigma-Aldrich; 2 mM L-glutamine, Thermo Fisher; 1 mM sodium pyruvate, Gibco; 50 µg/ml gentamicin, Sigma-Aldrich; 25 mM HEPES, Biochrom) with DEAE-dextran at a final concentration of 10 µg/ml and incubated for 2 days. Equal amounts of Luciferin-containing lysis buffer (10 mM MgCl₂, 0.3 mM ATP, 0.5 mM Coenzyme A, 17 mM IGEPAL (all Sigma-Aldrich), and 1 mM D-Luciferin (GoldBio) in 200 mM Tris-HCL pH 7.8) was added and after 2 min incubation samples were resuspended and luminescence was measured with a luminometer (Berthold TriStar² LB942). For IC₅₀ determination, the background signal (non-infected TZM-bl cells) was subtracted and IgG concentrations resulting in a 50% RLU reduction compared to untreated virus control wells were determined by using murine leukemia virus (MuLV)-pseudotyped virus as a control for unspecific activity. All samples were tested in duplicates.