ELISA and phage display - (Dec/03/2007 )
Hi, I have completed the phage display part of my experiment. I have recovered the peptides that seem to attach to my chosen target. I am now trying to perform an ELISA to verify that the peptides do infact bind to the target. I am coating my plates with the target and then adding biotin labelled peptides. Unfortunatly i am getting no significant readings. Can any one help with some hints and tips? or other methods that can verify binding of antigen to antibody? 
biotinylation successful?
Yes the phage born peptides were synthesised with a biotin label. I also have this concern: the target antigen which is coated on the plate is his tagged (is this a problem) then i add my biotin labeled phage peptides (antibody) then HRP-streptavidin is added. Result: no significant readings
usually one detects the binding phages using anti-M13 antibody. However, depending on the success of your panning sometime you can enrich non-binding phages resulting in no signal at all when testing on target by ELISA.
I'll suggest you include a positive control to confirm if this is not due to your reagents.
Yes the phage born peptides were synthesised with a biotin label. I also have this concern: the target antigen which is coated on the plate is his tagged (is this a problem) then i add my biotin labeled phage peptides (antibody) then HRP-streptavidin is added. Result: no significant readings
I'll suggest you include a positive control to confirm if this is not due to your reagents.
Yes the phage born peptides were synthesised with a biotin label. I also have this concern: the target antigen which is coated on the plate is his tagged (is this a problem) then i add my biotin labeled phage peptides (antibody) then HRP-streptavidin is added. Result: no significant readings
I dont use anti-M13 as the phage is not used in the elisa, just the peptide sequence synthesised from the phage end and biotinylated.
Sometimes you get background phage that actually don't bind to your target but bind to other molecules that also appear in your panning experiments. For example a colleague of mine told me that once he was working with a His-tagged target and using a Ni-affinity column and all he got out of the panning experiment were poly-His phage displayed peptides that bound to the column instead of the target.
Did you get any consensus sequence from the obtained phage displayed peptides? If so, where was the binding site located, N- or C-terminally. You could have blocked binding of your target to the peptides by biotinylation.
I hope it helps.
Miha
why don't you coat the wells of the ELISA plate with avidin (400ng/well), block with BSA 1%, capture your peptide (200ng/ml is enough) and after one hour of incubation wash the plate and use a solution of your selector molecule (100ng/well) and finally you use an antibody against the Fc region of your molecule (if is an antibody) or another tag.
I've done this procedure and really work. I hope you have lucky in this!!!
I've done this procedure and really work. I hope you have lucky in this!!!
Thanks i will give it a try