I am working on recombinant antibody technology, where phages are used for displaying antibody fragment on the surface. I am able to clone the antibody gene successfully in phagemid vector pSEX81, but at the time of ELISA panning, phages are not showing any binding with coated target antigen, although M13 expression suggests that phages are propagated successfully. Please advise.
The main query that I am facing is whether the vector that I am using is correctly displaying the gene of interest or not. For checking it, I have also propagated phages against some proteins for which we have antibodies to detect in ELISA. But, did not get positive result in that assay as well. It will be helpful if you could suggest any positive controls or any other suitable phagemid vector for scFV display. I'm stuck at this part of workflow.
Dear Swapnil,
which strain of bacteria do you use? I tried several of them, and it seems that the best yields of M13-antibody expression are achieved in the SURE strain (Stratagene, now Agilent). Please ask if you have more questions.
Yours Marcin
Dear Marcin, Thank you for your remark. Im using XL-1B MRF' strain. Basically I'm keeping M13 as a control and want to generate antibodies against different therapeutic proteins by infecting transformed XL-1B cells with Hyperphage M13. Im not observing display of my gene of interest on phages though.
Hi Swapnil,
pComb3XX vector works great. How are you detecting the phages in your ELISA?
Hi Daad Abi-Ghanem,
Thank you for your suggestion. Will check it out.
For ELISA, I am using Anti-M13 HRP conjugate for detecting M13 expression. Whereas, for probing phages displaying ScFv, I am coating target Ag on microtitre plate and using phages as primary antibody, detected by anti-M13 conjugate as detection Ab. I almost always get positive M13 expression, but protein binding is not seen.
Is the anti-M13 specific for the group 8 coat protein? (it needs to be). I use the one from GE Healthcare, it works really well.
Taking a step back, did you check your sequences after cloning? Is your library large enough? How are you performing the panning rounds? Check out "Phage Display: A laboratory manual", by Barbas et al., great reference book.
Good luck.
When we checked the sequence, it had shown only VH fragment as cloned. The main issue is we are not getting ScFv i.e VH + VL cloned in frame in the vector, which might be contributing to no binding with target Ag in ELISA.
I will again check the specificity of our anti-M13 for coat proteins. Further, I don't have access to that reference book. If you have a soft copy, that will be very helpful
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