I perform 5 biopannings rounds and then did ELISA with the 'soup of phages' from each circle (coating with specfic antybody, then phages and antiM13). Biopannings from 1-4 had really low OD but number 5 was very positive. Therefore I picked up and amplified single clones. Next I performed the same ELISA but in parallel I coated additional wells with 'nonspecific' antybody.
All clones were positive for both coatings.. (negative controls were ok). Do you have any idea what could be the reason? Do I need more biopannings rounds? Thank you in advance!
After 5 round of biopannings chances are that your phage output is dominated by a single phage (peptide or antibody display?), and it might also be the case that you have "pushed" the selection too much and obtained a non specific binder (again peptide or phage?) a "sticky" one. I really don't think that an extra round of selection will fix your problem.
First, you would need to make sure that the positive result against the unrelated antibody is not due to a problem in ELISA. Are you properly blocking the plate? Are you diluting the phages in blocking solution to avoid non-specific background? Is it necessary to dilute the phages even more to reduce background?
If you are totally sure that your phages are really sticky, then you should start selection again, but with more stringent blocking conditions in order to obtain only specific phages if they are there. Another selection round (after 5) is not likely to fix the problem.
Thank you for the answers! Im pretty sure that the problem is not due to ELISA (controls works perfectly good). May I just ask what do you mean by blocking conditions? Shortly, I have magnetic beads to which I bound aIgG and then IgG1 then I block for 2h with buffer which contains 0,5% BSA. Do you think that I should increase concentration?
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