Human il 6r

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.

pIL6RmAb expression was quantitated by ELISA that detected the assembled form of mAbs with both HC and LC, as described [28 (link)]. Briefly, plates were coated with a goat anti-human gamma HC antibody (Southern Biotech). After incubation with the plant protein extract, an HRP-conjugated anti-human-kappa LC antibody (Southern Biotech) was used for detection. A plant produced mAb with human IgG1 CH and kappa CL (E16) [28 (link)] was used as a reference standard.
The ELISA for measuring the binding of pIL6RmAb to IL-6R was performed as described [29 (link)]. Human IL-6R (2 µg/mL, Sino Biological) was immobilized on microtiter plates. An HRP-conjugated anti-human-gamma HC antibody was used as the detection antibody. The plates were developed with tetramethylbenzidine substrate (KPL Inc. Gaithersburg, MD, USA). A generic human IgG was used as an IgG isotype negative control. Experiments were performed at least two times with technical quadruplicate for each sample. The binding data were analyzed with GraphPad Prism software. KD was determined by non-linear regression analysis using a one-site binding model.