F8 maxisorp loose nunc immuno modules

Vaccine-induced Spike IgG was measured in serum samples by ELISA. 96-well plates (F8 MAXISORP LOOSE NUNC-IMMUNO MODULES, 469949, Thermo Scientific) were coated with 100 ng/well of SARS-CoV-2 (2019-nCOV) Spike S1 + S2 ECD-His-Recombinant Protein (40589-V08B1, Sino Biological) overnight at 4°C. Plates were washed three times with 1X PBS (5 min each time) and then blocked with blocking buffer [3% fetal bovine serum (FBS) in Dulbecco’s PBS (DPBS)] for 1 hour at room temperature, followed by washing and incubation at 4°C overnight with serially diluted serum samples (initial dilution, 1:20; 1:5 serial dilution) in blocking buffer at 100 μl per well. The following day, plates were rewashed and incubated with HRP-conjugated goat anti-hamster IgG (H+L) secondary antibody (HA6007; Invitrogen; 1:1500) for 1 hour at room temperature. After the final wash, plates were developed using TMB 1-Component Peroxidase Substrate (Thermo Fisher Scientific), followed by reaction termination using the TMB stop solution (Thermo Fisher Scientific). Plates were read at 450 nm wavelength within 10 minutes using a Microplate Reader (Bio-RAD).

To confirm the sensitization to OVA or ω5-gliadin, the plasma levels of IgE Abs specific to OVA or ω5-gliadin were determined using ELISA. Briefly, the wells of the F8 MaxiSorp Loose Nunc-Immuno™ Modules (Thermo Fisher Scientific) were coated with 100 μl of OVA (10 μg/ml) dissolved in phosphate buffered saline (PBS) or ω5-gliadin (20 μg/ml) dissolved in 0.1% acetic acid overnight at 4 °C. After washing with phosphate buffered saline containing 0.1% Tween 20 (PBS-T) six times, plates were incubated with 1% Block Ace® (DS Pharma Biomedical Osaka, Japan) for 2 h at room temperature. Then, 100 μl of each sample of rat plasma (diluted 1:10 in 1% Block Ace®) was added to each well and incubated for 2 h at room temperature. After washing with PBS-T, the wells were incubated with 100 μl of horseradish peroxidase (HRP)-conjugated mouse anti-rat IgE Ab (GeneTex, Irvine, CA, USA) (diluted 1:1000 in PBS) for 2 h at room temperature. The wells were washed with PBS-T and then incubated with 100 μl of 3,3′,5,5′-tetramethylbenzidine solution (KPL, Gaithersburg, MD, USA) at room temperature. After 15 min incubation, the reaction was terminated with 100 μl of 1 mol/l phosphoric acid. Absorbance was measured at 450 nm against 630 nm as a reference using a Multiskan GO (Thermo Fisher Scientific).