Hi Seyyed,

As Thamsanqa said before, make sure your immunotubes are correctly blocked to avoid non-specific binding– that may be the problem. It's ok to use immunotubes instead of ELISA plates, many people in my lab use them.

Anyway, I use Barbas's panning protocol and there is no need to use a control during the panning experiment. Normally, you will need 3-6 rounds of panning to enrich your library, and you should increase the stringency of the panning conditions in each round (by increasing the number of washes and reducing the amount of coated antigen) to allow the enrichment of specific binders. After several rounds of panning, you can test the phage resulting from each round by polyclonal ELISA, and it is at this moment when you can include your negative controls. Coat an ELISA plate with your antigen of interest and a control antigen and add phages pools from each round of panning (you can also add another phage, e.g. helper phage as a second negative control). Detect bound phage with an appropriate antibody (normally HRP-labelled anti-M13). With this ELISA you should be able to see if your panning worked and in which round you can find more specific binders. 

Hope this helped. Good luck with your experiments!

Hi Seyyed, Thamsanqa is correct. If your peptide is small then it will not present a surface large enough for the phage to bind when your peptide is bound to plastic.

You can get around that by immobilizing your peptide onto affinity resins that have spacer arms, like Affi-Gel.