Evaluating Atezolizumab Binding to PD-L1

The binding ability of plant-produced Atezolizumab to human PD-L1 was investigated in a functional antigen-binding ELISA. A 96-well, half-area microtiter plate (Corning, New York, USA) was coated with recombinant huPD-L1 protein (R&D System, Minneapolis, USA) overnight at 2 µg/ml in 1×PBS. Then, the plate was washed with 1×PBS-T and blocked with 5% (w/v) skim milk in 1×PBS. Plant-produced Atezolizumab, Tecentriq standard, and plant-produced Nivolumab control^{23 (link)} were serially diluted and incubated on the coated plate for 2 h at 37 °C. Detection was with goat anti-human Kappa-HRP (SouthernBiotech, Alabama, USA) at 1:3,000 in 1×PBS and peroxidase activity was determined by TMB one solution substrate (Promega, Wisconsin, USA). Color development was monitored and stopped with 1M H₂SO₄. The absorbance was measured at 450 nm on a NS-100 Nano Scan microplate reader (Hercuvan, Shah Alam. Malaysia).