Quantification of BaMV-based Protein Expression

For quantification of TP expression levels of different BaMV-based vectors, ELISA was performed as described previously (Jiang et al., 2019 (link)). TSP samples were prepared from inoculated leaves with 1:5 (w/v) ELSIA coating buffer (0.1 M carbonate/bicarbonate buffer, pH 9.6). Following centrifugation at 12,000 × g for 10 min, the supernatant was recovered and quantified for TSP using Bradford colorimetric assay (Sigma-Aldrich, St. Louis, MO, United States). The concentration of TSP in each sample was adjusted to approximately 5.5–6.5 mg/mL and 0.8–1.0 mg/mL in IC or AWF, respectively. The 96-well microtiter plates were coated with 5-fold serial dilutions of protein extracts from non-inoculated, inoculated leaves and purified plant-made SS^Ext mIFNγ(SP)₁₀ or purified mIFNγ protein derived from E. coli for standard curve and the positive control. The concentration of TP was determined by comparison with known amounts of the purified mIFNγ protein derived from E. coli. All measurements were performed in triplicates.