Temporal Expression Profile of mAb in N. benthamiana

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)].