Table 2. Results of indirect ELISA assays (absorbance values at 405 nm) and quick test strip to serological detection, respectively, of GFP and BAR proteins in leaf samples from co-transforming events of Setaria viridis. Primary antibody against the C-terminal portion was used for GFP detection, while for BAR protein detection was used the QuickStix™ Kit for PAT/bar (EnviroLogix, Inc., USA).
| Sample | Absorbance values at 405 nm | ELISA for GFP | Quick test strip for BAR |
|---|---|---|---|
| Buffer | 0.101 | Negative | Negative |
| SvA10.1 (negative control; wild-type plants)a | 0.332 | Negative | Negative |
| ECWT1 (GFP-positive control 1)b | 1.372 | Positive | Negative |
| ECWT2 (GFP positive control 2)c | 1.624 | Positive | Negative |
| EC1 | 0.762 | Positive | Positive |
| EC2 | 0.412 | Negative | Positive |
| EC3 | 0.358 | Negative | Positive |
| EC4 | 0.421 | Negative | Positive |
| EC5 | 0.456 | Negative | Positive |
| EC6 | 0.309 | Negative | Positive |
| EC7 | 0.695 | Positive | Positive |
| EC8 | 0.869 | Positive | Positive |
| EC9 | 0.987 | Positive | Positive |
| EC10 | 0.379 | Negative | Positive |
a=wild-type Setaria viridis A10.1 (non-transgenic; negative control for GFP in the indirect ELISA); b and c=transgenic plants positive control for ZmUBI-1:GFP and negative control for CRISPR/Cas9 NHEJ (used as positive controls for GFP protein in the indirect ELISA); EC1 to EC10=co-transforming events PCR-positive to ZmUBI-1:GFP and CRISPR/Cas9 NHEJ minimal expression cassette.