Binding Activity of Anti-CTLA-4 2C8 mAb

Recombinant human CTLA-4 protein (huCTLA-4) (11159-H08H, SinoBiological, China) and mouse CTLA-4 protein (muCTLA-4) (50503-M08H, SinoBiological, China) were used to verify the binding activity of plant-produced anti-CTLA-4 2C8 mAb to its target. Briefly, microtiter plates (3690, Corning, United States) were coated at a concentration of 2 µg/mL overnight at 4°C. Then, the plates were washed with 1X PBST and blocked with 5% (w/v) skim milk in 1X PBS for 1 h at 37°C. Two-fold serial dilutions starting from 8 µg/mL of plant-produced anti-CTLA-4 2C8 antibody or plant-produced irrelevant antibody (anti-PD-1) (Rattanapisit et al., 2019 (link)) or human IgG1 isotype control (ab206198, Abcam, United Kingdom) were added into the plates and incubated for 2 h at 37°C. After three-time washes with 1X PBST, the plates were incubated with peroxidase-conjugated goat anti-human kappa antibody (dilution 1:2500) (2060-05, SouthernBiotech, United States) for 1 h at 37°C. The plates were washed and then developed using TMB one solution substrate (Promega, United States) for 15 min at room temperature, before quenching with 1 M H₂SO₄. The absorbance was measured at 450 nm by a microplate reader (Hercuvan, Model: NS-100).