Salivary antibodies
Parents self-collected saliva in a 50 mL conical Falcon tube. Children
were provided a SalivaBio swab and Salimetrcs swab-storage tube.
Children produced between 0.1-1 mL of saliva from the swab, and parents
provided on average 2 mL. After centrifugation, saliva samples were
aliquoted and stored at −80 °C until analysis. Immuno MaxiSorp 96-well
ELISA plates (Thermo Fisher Scientific, USA) were coated overnight at
4 °C with 2 µg/mL recombinant SARS-CoV-2/2019-nCoV S1 protein (Sino
Biologicals) diluted in PBS. Wells were blocked with 10% skim milk in
PBST (PBS + 0.1% Tween 20) at room temperature for 1 h. Two-fold serial
dilutions of saliva samples in PBST were transferred to the ELISA plates
(in duplicate) and incubated at room temperature for 1 h. Saliva from an
asymptomatic individual confirmed negative for SARS-CoV-2 by clinical
testing was used as a negative control. Saliva from a convalescent
individual recently infected with SARS-CoV-2 was used as a positive
control and pre-COVID saliva samples were used as negative controls.
Antibody binding was detected with anti-human secretory IgA (sIgA,
1:10,000; Merck; followed by 1h incubation with biotinylated anti-mouse
IgG detection antibody, 1:1,000; Southern Biotech) and biotinylated IgG
(1:10,000; Assay Matrix) for 1 h at room temperature, then
Streptavidin-HRP (1:5000; Life Technologies) in PBST for 45 min at room
temperature. Colour was developed with TMB solution (Sigma-Aldrich) and
H2O2 with the reaction stopped using 2 M
H2SO4. Absorbance at 450 nm was read on
a microplate reader and used to calculate end point titres of samples.
Cut-off values for each antibody class was defined as two standard
deviations above the maximum titre from the corresponding negative
controls.