Costar high bind flat bottom plates

Plasma was assessed for anti-RBD IgG and IgA by ELISA. 96-well ELISA plates (Costar high-bind flat-bottom plates, Corning) were coated with 10 μg/mL RBD in carbonate buffer (50 mM sodium carbonate/sodium bicarbonate, pH 9.6) overnight at 4 °C. The following day, plates were washed three times in PBS with 0.05% Tween 20 (PBS-T) and then blocked with 1x casein (Sigma) for 1 h at RT. Following blocking, wells were washed three times with PBS-T and further incubated with six 10-fold dilutions of plasma in 1x casein for 2 h at RT. Wells were then washed five times with PBS-T and incubated for an additional hour at RT with horseradish peroxide (HRP)-conjugated antibodies against mouse IgG, IgG1, IgG2b, IgG3, or IgA (Southern Biotech). After five washes with PBS-T, bound RBD-specific Ig was detected with tetramethylbenzidine (TMB) substrate. Stop solution (3% H₂SO₄ + 1% HCl) was added after 18 min of TMB incubation at RT, and the OD was measured at 450 and 570 nm on an Epoch Microplate Spectrophotometer (BioTek). Background signal at 570 nm was subtracted from the OD at 450 nm. Fold-change over the average of blank wells was then calculated and log-transformed. The area under the curve (AUC) of log-transformed fold change versus log-transformed dilution was then calculated.

mMCPT-1 in the post-challenge serum was measured using the MCPT-1 mouse uncoated ELISA kit (ThermoFisher) following the manufacturer’s protocol. For the BLG-specific IgE ELISA, serum from the sensitized mice was diluted and added to BLG-coated 96-well ELISA plates (Costar highbind flat-bottom plates, Corning). BLG-specific IgE Abs were detected with goat anti-mouse IgE-unlabeled (Southern Biotechnology Associates, Birmingham, AL) and rabbit anti-goat IgG-alkaline phosphatase (Invitrogen, Eugene, Oregon) and developed with p-nitrophenyl phosphate “PNPP” (SeraCare Life Sciences, Inc. Milford, MA). For the BLG-specific IgG, IgG1, and IgG2a ELISA, serum from the sensitized mice was diluted and added to BLG-coated 96-well ELISA plates. BLG-specific IgG,IgG1, and IgG2a was detected using goat anti-mouse IgG-HRP, IgG1-HRP, or IgG2a-HRP conjugated (Southern Biotechnology Associates) and TMB liquid substrate system for ELISA (Sigma-Aldrich, St. Louis, MO). The plates were read in an ELISA plate reader at 405 nM (IgE) or 450 nm (IgG, IgG1, and IgG2a). OD values were converted to nanograms per milliliter of IgE, IgG, IgG1, or IgG2a by comparison with standard curves of purified IgE, IgG, IgG1, or IgG2a.