Fig. 1.

Expression of N6 antibody in N. benthamiana. A Human IgG kappa (HuIgGk) from human serum (Sigma), mock-infiltrated sample from leaf disk (mock), N6 extracted from leaf disk (pN6), and Precision Plus Protein™ All Blue Pre-stained Protein Standards (M) were run, either reduced (R) or not reduced (NR), on SDS-PAGE before blotting onto nitrocellulose. Heavy chain was detected with mouse anti-human IgG Fc domain and light chain was detected with goat anti-human kappa light chain. Secondary antibodies were donkey anti-mouse with green fluorescent tag, and donkey anti-goat with red fluorescent tag. Black arrows indicate, from top to bottom, fully assembled antibody (yellow), heavy chain (green) and light chain (red). B Sandwich ELISA detecting fully assembled antibody in plant crude extract. Leaf disks taken from N. benthamiana transiently expressing N6 (triangle), human IgG kappa positive control (circle) or plants mock-infiltrated with infiltration solution only (square) were extracted in PBS and introduced to an ELISA plate coated with goat anti-human IgG Fc domain antibody. Bound antibodies were detected using HRP-conjugated goat anti-human IgG kappa light chain antibody. Representative of 3 biological replicates (i.e. separate plants and infiltration experiments). Each ELISA was performed with 2 technical replicates. Means derived from 2 leaf disks per sample ± S.D. Yields were estimated using Graphpad Prism software, fitting to Michaelis Menton equation