I previously developed a mouse single chain variable fragment (scFv) and characterized it after cloning into a human IgG1 scaffold. Had great potency and selectivity in vitro and in vivo. I now want to test the scFv in more scaffolds to determine other applications for the scFv. I have tried cloned the heavy and light chains into rabbit or mouse IgG vectors and expressed in HEK293T cells. However, I am not seeing binding with ELISA or flow cytometry. All vectors and inserts have been verified via sequencing. Any idea what is happening?
Were you able to confirm that the corresponding proteins are actually produced?
Annemarie Honegger Yes, I ran the purified protein on a gel under reduced and non-reduced conditions and saw the appropriate heavy and light chain bands.
Are you using directly conjugated antibody for your binding experiments or could it be a problem with the respective detection antibodies?
If you pm me the protein sequences, I can check if there is any problem with the sequences (both the functional and the non-functional constructs)
- Badges
- Science topic
- Similar topics
- Correction