Hrp conjugated anti human kappa lc antibody

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.

N. benthamiana leaves agroinfiltrated by ZV1 LC and HC expression vectors were harvested 5, 6, 7, and 8 days post agroinfiltration (DPI) to evaluate the temporal expression profile of the mAb by using an ELISA that detected only the assembled form of mAbs as we previously described [29 (link)]. Briefly, total leaf soluble protein was obtained by homogenizing leaves in extraction buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl) and clarified by centrifugation at 15,000× g for 30 min at 4 °C. Microtiter plates were coated with a goat anti-human gamma HC antibody (Southern Biotech, Birmingham, AL, USA) and incubated with the plant protein extract. After incubation and washing, an HRP-conjugated anti-human-kappa LC antibody (Southern Biotech, Birmingham, AL, USA) was used for detection. A plant-produced IgG isotype control mAb (E16) was used as a reference standard. For functional evaluation experiments, larger-scale ZV1 variants were extracted from leaves at 8 DPI as described above and purified with a Protein A-based method we developed previously [30 (link)].