Hello everyone.

I am a beginner in phage display of antibodies. We have an in-home prepared phage display antibody library. I performed biopanning against an antigen, a purified protein, with phage expressing the library. In order to make the selection stringent, I performed 60x washings 1minute each (30x with PBS-T 0.1% tween and 30x with PBS).

After biopanning, when comparing to number of eluted phages in Antigen coated vs No Antigen wells, I got ~70 fold more colonies in Antigen coated wells compared to No Antigen well.

{Short Summary}

When I performed monoclonal phage ELISA, I could not observe any difference between No-Ag vs Ag well for each clone. Infact, in some cases the OD was higher in No-Ag as compared to Ag. Seeing that the enrichment at biopanning level was nice, it is really disheartening to see none of the clones reacted. What could be the possible reasons ? Could it be that the antibodies might cross react with blocking solution components ? Or is it that none of the clone binds ?

{Detailed Procedure}

Therefore, I proceeded with colonies that have correct antibody fragment (based on colony PCR with vector specific primers) and performed a monoclonal phage ELISA. For monoclonal phage ELISA, 1mL secondary culture was set up from primary culture of each colony. The secondary culture was then infected with helper phages OD600=0.5 at 1:20 infection ratio and then Kan(helper phage) and IPTG were added. This culture was then grown @ 37degC under shaking for 9-10 hours. Phages were then purified with PEG/NaCl and then used for phage ELISA.

For each clone, 3wells were No-Ag while 3-wells were Ag coated. Purified phages were diluted in blocking solution and divided equally in all 6-wells, following blocking the wells. Phages were detected with anti-M13KO7 Hyper Immune Serum and further detected with anti-Mouse AP conjugated antibody.

I could not observe any difference between No-Ag vs Ag well for each clone. Infact, in some cases the OD was higher in No-Ag as compared to Ag.