Mtp 900lab

The binding of TGF-βRII-Ig to TGF-β1 was evaluated by ELISA. A 96-well ELISA microplate (SUMITOMO BAKELITE, Akita, Japan) was coated with 10 μg/mL of TGF-βRII-Ig or Control IgG-B diluted in PBS at 37°C overnight. After washing with PBS containing 0.05% Tween20 (KANTO KAGAKU, Tokyo, Japan), the plate was blocked with Super Block (Thermo Fisher Scientific) at 37°C for 1 h. Recombinant human TGF-β1 (240-B/CF, R&D systems) was added to the plate at various concentrations (4-fold dilution from 8 to 0.125 ng/mL), followed by incubation at 37°C for 1 h. Then, 1 μg/mL biotinylated chicken anti-human TGF-β1 antibody (R&D systems) was added and incubated at 37°C for 1 h. The reaction was developed using peroxidase-conjugated NeutrAvidin protein (Thermo Fisher Scientific) and TMB One Component Substrate (Bethyl Laboratories, Montgomery, TX, USA) and then stopped with 0.18 M H₂SO₄. As a dilution buffer for recombinant TGF-β1, anti-TGF-β1, and NeutrAvidin, PBS containing 1% bovine serum albumin was used. The absorbance was measured at 450 nm with the MTP-900Lab (CORONA ELECTRIC).

A 96-well ELISA microplate (Nunc maxisorp, Thermo Fisher Scientific) was coated with 10 μg/ml of dog IgG1 antibody (A40-120A, Bethyl Laboratories) targeting canine IgG-A and IgG-D or dog IgG2 antibody (A40-121A, Bethyl Laboratories) targeting canine IgG-B, both diluted in PBS at RT for 1 h. After washing with PBS containing 0.05% Tween20 (Kanto Chemical), the plate was blocked with PBS containing 0.05% Tween20 (Kanto Chemical) and 1% bovine serum albumin (BSA) (Thermo Fisher Scientific) (1% BSA-PBS-T) at 37°C for 1 h. Samples were prepared at 1:2,000 dilution, and added to the plate, followed by incubation at RT for 1 h. The reaction was developed using HRP-conjugated dog IgG1 antibody (A40-120P, Bethyl Laboratories) or HRP-conjugated dog IgG2 (A40-121P, Bethyl Laboratories) and TMB One Component Substrate (Bethyl Laboratories) and then stopped with 0.18 M H₂SO₄. We used 1% BSA-PBS-T as a dilution buffer for the collected samples and the detection antibody. The absorbance was measured at 450 nm with the MTP-900Lab multiplate reader (CORONA ELECTRIC).

Canine PBMCs from healthy beagles were cultured with SEB at 1 μg/mL for 72 h. Recombinant human TGF-β1 (0.78 nM) alone or in combination with TGF-βRII-Ig (78 nM) were added to the medium. Control IgG-B was used as negative control for TGF-βRII-Ig. IL-2, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α concentrations in the culture supernatants were measured by Canine IL-2 DuoSet ELISA (DY1815, R&D systems), Canine IFN-γ DuoSet ELISA (DY781B, R&D systems), and Canine TNF-α DuoSet ELISA (DY1507, R&D systems), respectively. The absorbance was measured at 450 nm with the MTP-900Lab (CORONA ELECTRIC).