Measurement of Anti-spike IgG Titers

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.