SARS-CoV-2 Spike Protein ELISA Protocol

ELISA experiments were carried out as previously described^{41 (link)}. Briefly, Nunc Maxisorp ELISA plates (ThermoFisher) were coated with 100 ng/well of stabilized soluble SARS-CoV-2 spike protein^{24 (link)} (with His-tag cleaved to remove potential cross-reactivity) in 1X PBS at 4 °C for 16 hr. To eliminate fold-on-specific binding, 50 μg/mL of fold-on protein was added to serial dilutions of heat-inactivated sera for 1 hr at room temperature (RT). After blocking in PBS-Tween (PBST) supplemented with 5% nonfat milk, plates were incubated with sera for 1 hr at RT. After blocking in PBS-Tween (PBST) supplemented with 5% nonfat milk, plates were incubated with serial dilutions of heat-inactivated sera for 1 hr at RT. Secondary antibody, goat anti-mouse IgG conjugated to horseradish peroxidase (ThermoFisher), was then added, followed by excitation with 3,5,3′5′-tetramethylbenzidine substrate (KPL). Each step in this procedure was followed by standard washes in PBST. Endpoint titers were calculated as the dilution factor that resulted in an optical density exceeding 4X background (secondary antibody alone).