3 3 5 5 tetramethylbenzidine solution

Blood samples were taken from the sheep. An enzyme-linked immunosorbent assay was used to measure the specific serum antibody concentrations (IgM and IgG) against α-Gal in the sheep. Microtiter plates were coated with 100 μL per well of bovine serum albumin (BSA) conjugating synthesized α-Gal (α-Gal-BSA; Dextra Laboratories, Reading, United Kingdom) in PBS (pH 7.4) (at 1 μg/mL for the IgM and IgG isotypes) and incubated at 37°C for 1 hours. Then, the plates were washed with PBS containing 0.05% (v/v) Tween 20. Sheep sera (100 μL per well) were added to the α-Gal-BSA–immobilized wells in BSA-Tween 20 (PBS, pH 7.4, 3% BSA, 0.01% Tween 20), and the plates were incubated for 1 hour at 37°C. Horseradish peroxidase-conjugated rabbit anti-sheep IgM and IgG (AbD SeroTec, Oxford, United Kingdom) were used as the secondary antibody (1:10,000) at the IgM and IgG dilution in BSA-Tween 20. A color reaction was developed with 3,3′,5,5′-tetramethylbenzidine solution (BD Biosciences, San Diego, Calif). Absorbance was measured at 450 nm in an enzyme-linked immunosorbent assay reader.

To this end, 96-well Nunc plates (Thermo Fisher Scientific) were coated with 100 ng/well RID, an RID-fused protein, or 10⁶ PFU/well PR8 (H1N1) virus and were incubated at 4°C overnight. The plates were washed with PBST and were blocked with 1% BSA in PBST for 1 h at room temperature. Next, 100 μl serum samples, at various dilutions, was added into each well, and the plates were incubated for 1 h at room temperature, followed by washing with PBST. One hundred microliters of a secondary goat anti-rabbit IgG antibody or goat anti-mouse IgG antibody conjugated with HRP (Sigma-Aldrich) was added into each well at a dilution of 1:10,000 and was incubated for 1 h. After a wash with PBST, 100 μl of the substrate 3,3′,5,5′-tetramethylbenzidine solution (BD Biosciences) was added into each well, and then the plate was developed in the dark for 30 min. The colorimetric reaction (blue to yellow) was stopped by addition of 2 N H₂SO₄ 50 μl/well, and OD₄₅₀ was measured on a microplate reader (FLUOstar Optima; BMG Labtech).