Elisa coating buffer

IL-10 levels in cell culture supernatants were measured using the ELISA MAX™ Standard Set Human IL-10 (BioLegend) or Human IL-10 ELISA Set (Diaclone), according to the manufacturer’s instructions, using ELISA Coating Buffer (BioLegend). IL-13 in cell culture supernatants was measured using the Human IL-13 ELISA Development Kit (HRP) (Mabtech) or Human IL-13 DuoSet ELISA (R&D Systems) according to the manufacturer’s instructions. For IL-17A measurement Human IL-17A ELISA Development Kit (HRP) (Mabtech) was used, according to the manufacturer’s instructions. Nunc-Immuno™ MicroWell™ 96 well plates (Sigma-Aldrich) were used. Plates were developed using the TMB Substrate Set (BioLegend), with H₂SO₄ added to stop the reaction. Absorbance was read at 450 nm and 570 nm wavelengths using an Epoch 2 Microplate Spectrophotometer (BioTek) or an Asys UVM340 microplate spectrophotometer (Biochrom). The absorbance at 570 nm was subtracted from that at 450 nm, and the concentrations were calculated based on standard curve equations.

Allergen-specific IgE antibody determination was performed as previously described (11 (link)). Briefly, 96-well microplates were immobilized with 5 µg/10 mL (plate) of recombinant Art v 1 protein on an ELISA Coating Buffer (BioLegend) overnight. On the following day, ELISA Assay Diluent (BioLegend) in phosphate-buffered saline (PBS) was added into the plates at 200 μL/well and incubated on a PST-60HL thermal shaker (BIOSAN, Latvia) for 1 h at room temperature (RT). The plates were then washed four times with ELISA Wash Buffer (BioLegend). The mouse serum samples were diluted in a 1:5 ratio with ELISA Assay Diluent and 100 µl were added to the wells and incubated for 1.5-2 h at RT with stirring. After washing (4x), anti-mouse biotinylated detection antibodies for IgE (1:200, Cat # 406904, BioLegend, 100 µl/well) were added and the plates were incubated for 1 h at RT with stirring. After additional washing (4x), plates were incubated with HRP Streptavidin (BioLegend, 1:1000, 100 µL/well) for 30 min at RT with stirring. After that, the washing (5x) was repeated, and on completion of which the TMB substrate (BioLegend, 100 µl/well) was added. The color reaction was stopped by adding a stop solution (100 µL/well), and the optical density was measured at 450 nm on a Stat Fax 2100 analyzer (Awareness Tech).

Corning ELISA plate wells were coated with 100µL of 100 μg/mL FCoV2-WV antigen or 100 μg/mL SCoV2 RBD antigen in sodium bicarbonate ELISA coating buffer at pH 9.5 (BioLegend, San Diego, CA, USA) and incubated overnight at RT. (Due to the limited amount and frequency of serum collection during COVID-19 between 25 March 2020 and 4 January 2021, ELISA with FCoV RBD antigen was not performed. Instead, more specific immunoblot analyses, using FCoV1 or FCoV2 RBD antigen, were conducted.) The next day, the plates were washed three times with phosphate-buffered saline tween (PBST). Non-specific binding sites were blocked with 100 µL per well of blocking solution (5% non-fat dry milk in sterile PBST- 0.5% Tween-20) for 1 h at 37 °C. After washing with PBST three times, 10 µL of cat serum was diluted in 0.990 mL of blocking solution (1:100) and incubated at RT overnight. After washing, horseradish peroxidase (HRP)-conjugated goat anti-cat IgG diluted 1:4000 (SouthernBioTech, Birmingham, AL, USA) in PBST was added and incubated at RT for 2 h. After washing, 100 µL of 3,3,5,5-tetramethylbenzidine High Sensitivity Substrate Solution (BioLegend) was added to the wells and incubated at RT for 15 min. The reaction was stopped by adding 100 µL of 1 N HCL in sterile water. The ELISA titer was measured at OD450 using BioTek’s Synergy HTX Multi-Mode Microplate Reader (BioTeK, Winooski, VT, USA).