One possibility might be that you have a low-affinity binder. On the phage, the multivalent display may be just sufficient to get a positive signal, while in monomeric form you may lose your antibody during ELISA incubation and washing steps.

In my experience its quite common to have good phage-displayed binders that are not so good (or totally negative) when produced as soluble proteins in periplasmic space, due to frequent aggregation and missfolding problems. Have you produced the soluble protein in periplasm? If so, sometimes mammalian cell expression of the soluble molecules overcomes that problem.

Gertrudis: Valencio specifically said that he performed the ELISA with purified protein after IMAC and Gel filtration, therefore his problem is not due to protein production.

Thank you Annemarie and Gertrudis for your valuable insights. The protein was purified from E.coli by osmotic shock method after one freeze thaw cycle. We recovered around 0.5 mg from a 400 ml culture after IMAC.

I have observed protein production problems after obtaining the binders outside the phage context, but also that binders (even those produced) are no longer active.

Hi there, I found your discussion about phage display.

I would like to know, please, if you have any experience with the T7 phage since I have to start using it for nanobodies expression but I have never worked with it.

Any advice is more than welcome.