Plasmas were isolated by Ficoll density gradient from blood samples. Plasmas were heat-inactivated for 1 h at 56 °C and stored at −80 °C until use. We used plasma samples from uninfected/unvaccinated donors collected before the pandemic as negative controls in ELISA, ADCC, and cytometry assays, and to calculate the seropositivity threshold. The CR3022 monoclonal antibody (mAb) (a receptor-binding domain (RBD)-specific monoclonal antibody) and the CV3-25 mAb (a conformationally independent S2-specific mAb) were used as positive controls in ELISA using the ancestral RBD and flow cytometry assays, respectively [10 (link),30 (link),31 (link),32 (link)]. We used as secondary antibodies (Abs) a Horseradish peroxidase (HRP)-conjugated Abs that detect the Fc region of human IgG (Invitrogen) in ELISA assays and Alexa Fluor-647-conjugated goat antihuman antibodies detecting all Ig isotypes (antihuman IgM, IgG, IgA; Jackson ImmunoResearch Laboratories, cat # 109-605-064) in flow cytometry experiments.
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