96 well high binding elisa plates

Anti-spike IgA titers were also measured by indirect ELISA. Specifically, 96-well high-binding ELISA plates (Thermo Fisher Scientific) were coated overnight at 4 °C with 100 μL of 0.8 μg/mL (80 ng/well) of Spike protein diluted in PBS. After washing plates with PBS-T, wells were blocked with 200 μL 3% Skim milk (Thermo Fisher Scientific) in PBS and incubated at 37 °C for 2 h and washed with PBS-T. 100 μL of sample was loaded on the plates using half-log serial dilution (equivalent to 3.162 × serial dilution starting at 1:10) and incubated at 37 °C for 1 h. Plates were washed again with PBS-T and 100 μL of goat anti-mouse IgA-HRP (Abcam, Cambridge, UK) diluted 1:10,000 added to each well. Plates were incubated at 37 °C for 45 min and washed with PBS-T. 100 μL of TMB substrate Sure-blue reserve one-component (Mandel Scientific Company Inc., Guelph, ON, Canada) was then added to each well and reaction stopped after 10 min incubation at room temperature by adding 100 μL of TMB stop solution one-component (Mandel Scientific Company Inc., Guelph, ON, Canada). Bound IgA Abs were detected spectrophotometrically at 450 nm, and IgA antibody titers were determined as above for IgG.

Anti-spike total IgG titers in serum were measured by indirect ELISA with SmT1-R, -B, or -D as previously described^{10 (link)}. Briefly, 96–well high-binding ELISA plates (Thermo Fisher Scientific) were coated with 0.3 µg/mL SmT1 protein diluted in PBS. Serum samples were serially diluted 3.162-fold and added to the plates to allow for binding of antibodies to the protein. Bound IgG was detected with goat anti-mouse IgG -HRP (1:4000, Southern Biotech, Birmingham, AL, USA) prior to the addition of the substrate o-phenylenediamine dihydrochloride (Sigma-Aldrich). Bound IgG Abs were detected spectrophotometrically at 450 nm. Titers for IgG in serum were defined as the dilution that resulted in an absorbance value (OD₄₅₀) of 0.2 and was calculated using XLfit software (ID Business Solutions, Guildford, UK). Samples that did not reach the target OD were assigned the value of the lowest tested dilution (i.e., 100) for analysis purposes. No detectable titers were measured in serum samples from naïve control animals.