Peroxidase goat anti human igg

For detecting anti-PD-1 and anti-CTLA-4 minibodies, T cells were transduced and maintained as described above, between 1.0–2.0 × 10⁶ cells/mL. 70 mL supernatant from day 11 of T cell expansion in vitro was collected and concentrated with Centricon Plus-70, as per the manufacturer’s instructions. A standard direct ELISA was performed with DuoSet Ancillary Reagent Kit 2 (R&D systems). After coating with recombinant human PD-1 and CTLA-4 protein (Abcam), a 96-well plate was loaded with the concentrated supernatants followed by peroxidase goat anti-human IgG (Jackson ImmunoResearch Laboratories) detection antibody. For detecting canine IFNγ, supernatant was collected from canine T cell and target cell 16-hr co-culture at 1:1 ratio. The detection was performed with canine IFN-gamma DuoSet ELISA kit (R&D Systems) as the introduction indicated.

Briefly, HEK cells were plated in a 100-mm-diameter dish and transfected with a plasmid coding for the target antigen to be confirmed as described above. At 36–48 hours post-transfection, cells were washed with cold PBS, and proteins were extracted in lysis buffer (50 mM Tris-HCl [tris-aminomethane hydrochloride] pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40 [nonyl phenoxypolyethoxylethanol], 0.1% sodium dodecyl sulfate [SDS], protease inhibitors, and Benzonase) for 30 minutes at 4°C. The extract was cleared by centrifugation (10,000g, 20 minutes), denatured in Laemmli buffer (62.5 mM Tris-HCl, 2% SDS, 10% glycerol, 100 mM DTT, 0.01% bromophenol blue), subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) separation, and then transferred on a polyvinylidene difluoride membrane using a semi-dry Western blot apparatus. Serum (diluted 1/100) was incubated overnight with the membrane. Antibody fixation was visualized using peroxidase goat anti-human IgG (Jackson ImmunoResearch, Cambridgeshire, United Kingdom) and chromogenic substrate (Fast 3,3′ Diaminobenzidine tablet, Sigma-Aldrich). A sample was positive if it bound to the recombinant protein on the membrane. A positive and a negative control were included in each experiment.