Human igg1

ELISA to examine the binding of mAb B38 and H4 to SARS-CoV-2 RBD protein was performed. Three biological replicates were performed, and each sample was subjected to three technical replicates to assess the mAb binding efficiency. Briefly, RBD of SARS-CoV-2 protein (His tagged RBD produced in Sf9 insect cells, Z03479; Genscript Biotech, United States) was bound to 96-well microplates (Greiner Bio-One GmbH, Frickenhausen, Germany). After overnight incubation, the plates were blocked with 5% skim milk (BD, Franklin Lakes, NJ, United States) in 1 × PBS for 2 h, washed three times with PBST, and incubated with plant-produced mAbs B38 and H4. After 2-h incubation at 37°C, sheep anti-human kappa LC conjugated with HRP (Southern Biotech, United States) at a dilution of 1:1,000 in 1 × PBS was added, and samples were incubated for 1 h at 37°C. The plate was then washed three times with PBST, developed using TMB substrate (Promega, United States), and the absorbance was read at 450 nm. The commercially available human IgG1 (Abcam, United Kingdom) and plant-produced human anti-PD1 antibody (Rattanapisit et al., 2019b (link)) was used as negative controls.

The plant-produced anti-PD-L1 antibody was quantified by sandwich enzyme-linked immunosorbent assay (ELISA). Briefly, 96-well plates (Greiner Bio-One, Kremsmunster, Austria) were coated with 50 μL of 1:1,000 dilution of goat anti-human IgG and were incubated overnight at 4°C. Then the plates were washed and blocked with 5% skim milk in PBS for 2 h at 37°C. The samples were diluted in a range of 1:1,500 to 1:100,000 for optimization and the quantification of purified antibody. 50 μL of diluted samples were added into each well and incubated for 2 h at 37°C, where human IgG₁ (Abcam, Cambridge, UK) was used as control to generate the standard curve. A 1:1,000 dilution of sheep anti-human kappa-HRP (The binding site, Birmingham, UK) was added and incubated for 1 h at 37°C after washing with PBST. The plates were developed by adding 50 μL of 3,3’,5,5’-Tetramethylbenzidine (TMB) substrate (Promega, US), followed by 1M H₂SO₄ for terminating the reaction. Finally, the plates were measured at an OD of 450 nm by using SpectraMax M5 microplate reader.