Evaluating Plant-Produced Anti-hIL-6R Antibody

Plant-produced anti-hIL-6R antibody binding activity was investigated by ELISA using specific human IL-6 receptor (hIL-6R) recombinant protein. Three biological replicates were performed. Each 96 half well in the microplate was coated with 25 µL of 2 µg/mL human IL-6R (Sino Biological, USA) and incubated at 4 °C overnight. The incubated plate was washed with 1X PBS-T (3 times) and blocked with 5%(w/v) non-fat milk in 1X PBS at 37 °C for 2 h. The plate was further incubated with plant-produced anti-hIL-6R samples at 37 °C for 2 h. After washing, the plate was treated with goat anti-human kappa-HRP (1:2,500) (Southern Biotech, Birmingham, USA) for 1 h at 37 °C. Finally, the plate was washed and incubated with 25 µL/well of 3,3′,5,5′-tetramethylbenzidine (TMB) (Promega®, USA). The reactions were stopped by addition of 25 µL/well of 1 M H₂SO₄ and the absorbance was read at 450 nm by microplate reader (Hercuvan system, UK). Plant-produced H4 mAb^{35 (link)} was used as negative control. The data were analyzed by GraphPad Prism 9.3 software (San Diego, CA, USA). The dissociation constant (K_D) was determined by non-linear regression analysis using a one-site binding model.