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.