Hello, i screened some clones in phage display strategy after 4 rounds of biopanning, which they were positive in both monoclonal and competitive phage ELISA, but after transforming into the HB2151 strain, the soluble scFvs with high level of expression couldn't bind to the antigen or they have very low affinity.
what should i do?
Thank you
Hi Reza,
First, after transforming into HB2151, did you confirm scFv expression by running a Coomassie SDS-PAGE gel and/or a Western blot? It's important to verify that the scFv is being properly produced and is soluble.
Second, which phage display format are you using? Does your phagemid encode an scFv–amber–pIII construct? If so, during phage ELISA, you'd have mainly scFv-pIII fusion proteins on the phage (multivalent display), while the soluble fraction would be only ~10% due to amber suppression inefficiency. That can lead to a misleadingly strong signal in ELISA due to avidity effects.
If you used hyperphage for packaging, it further increases valency, enhancing binding via avidity. However, when switching to helper phage, display becomes monovalent, revealing the actual (often lower) affinity of your clones. That could explain the drop in binding when switching to soluble scFvs.
Finally, I’d recommend checking not just for expression but also for proper folding and solubility of your scFv in HB2151. Poor expression or aggregation could easily explain the lack of binding.
Hope this helps!
Best wishes
Stöpa
Thank you for your guidance, Mr. Fortunately, I've checked the time-pointing expression and solved this problem.
- Badges
- Science topic
- Similar topics
- Mathematics
- Algebra
- Matrix