Serological Immunity
Plasma S1 and RBD ELISA
The ELISA method used to measure IgG, IgM, and IgA levels to SARS-COV-2 S1 and RBD protein was based on the Mount Sinai Laboratory method previously described Briefly, 96-well high-binding plates were coated with receptor binding domain (RBD) or S1 (Sino Biological, China) antigen diluted in PBS at 2 µg/mL. Serum samples were first screened with RBD antigen, and potential seropositive samples were then confirmed with S1 antigen. Goat anti-human IgG- (1:10,000) horseradish peroxidase (HRP) conjugated secondary antibody was used, and the plates were developed using 3.3’, 5.5’-tetramethylbenzidine substrate solution. Seropositive samples were titrated and calculated based on a World Health Organization (WHO) SARS-CoV-2 pooled serum standard (National Institute of Biological Standards and Controls, United Kingdom). Results were reported in International Units/mL. The cut-off for seropositivity was 8.36 IU/mL based on pre-pandemic samples, while seronegative samples were given half of the seropositive cut-off value.
Coronavirus antibody multiplex assay (blood and saliva)
A novel coronavirus multiplex bead array was designed as previously described19 consisting of SARS-CoV-2 spike 1 (Sino Biological), spike 2 (ACRO Biosystems), spike trimer (kind gift from Adam Wheatley), RBD (BEI Resources) and nucleoprotein (ACRO Biosystems). Tetanus toxoid (Sigma-Aldrich), influenza hemagglutinin (H1Cal2009; Sino Biological) and SIV gp120 (Sino Biological) were also included in the assay as positive and negative controls respectively. Antigens were covalently coupled to magnetic carboxylated beads (Bio Rad) using a two-step carbodiimide reaction and blocked with 0.1% BSA, before being resuspended and stored in PBS 0.05% sodium azide till use.
The antigen-coupled beads were combined to form a coronavirus multiplex bead cocktail to investigate serological signatures from plasma and saliva samples. Briefly, 20 µL of working bead mixture (1000 beads per bead region) and 20 µL of diluted plasma (final dilution 1:200) or 20 µL of diluted saliva (final dilution 1:50) were added per well in 384 well plates and incubated overnight at 4°C on a shaker. Fourteen different Fc detectors were used to assess coronavirus-specific antibodies as previously described35, including phycoerythrin (PE)-conjugated mouse anti-human pan-IgG, IgG1-4 and IgA1-2 (Southern Biotech; 1.3 µg/mL, 25 µL/well). IgM (biotinylated mouse anti-human IgM (mab MT22; Mabtech; 1.3 µg/mL, 25 µL/well), C1q protein (MP Biomedicals) and FcγR dimers (higher affinity polymorphisms FcγRIIa-H131, lower affinity polymorphisms FcγRIIa-R131, FcγRIIb, higher affinity polymorphisms FcγRIIIa-V158, lower affinity polymorphisms FcγRIIIa-F158; 1.3 µg/mL, 25 µL/well; kind gifts from Bruce Wines and Mark Hogarth36 were first added to the beads, washed, and followed by the addition of PE-conjugated streptavidin (1.3 µg/mL, 25 µLµ/well). Assays were read on a Flexmap 3D with x-PONENT 4.2 software and performed in duplicate. Antibody levels are reported as median fluorescent intensity (MFI) of the PE signal associated with each bead. Pre-SARS-CoV-2 pandemic samples were used as controls in multiplex assay. SARS-CoV-2 plasma antibodies (positive/negative) and salivary antibodies (positive/negative) cut-off thresholds were determined by calculating the average plus two-standard deviations of respective pre-pandemic control data.