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.