problems with phage display systems
jmajor at blkbox.COM
Sat Jun 25 23:37:16 EST 1994
I hope others out there have used the phage display system.
We have been using the Pharmacia phage display system and have
encountered problems. If anyone has any advice with the following I
would be very interested to know.
We have cloned an antibody into the scFv system for subsequent
mutagenic library production and panning. The orginal scFc binds
reasonably well to the peptide antigen used for ELISA and panning on
Immulon II plates. Expected complexity of the library is about
1 x 10e5. It appears the library is pretty complete. Nevertheless,
using several different panning methods we are unable to enrich for
specific clones which bind above background control peptide. Have you
had experience with panning like this and what conditions would you
recommend for the binding/washing? We use 5ug/ml of peptide on the plate
to coat and block with 2% BLOTTO. We incubate phage on coated washed
plates for a couple of hours and wash a variable length of time-- up to
10-20 times. I don't see any real enrichment and the subclones examined
by ELISA indicate this as well.
I have found that when one examines the panned amplified mixtures
along the way that the extent of non-specific binding increases with each
panning. Moreover, on ELISA I can see that this non-specific binding
appears to be due at least in part on binding to the plastic itself, since
there is high signal when one just uses BLOTTO coated plates with on
peptide. We use Pharmacia recommended 2% BLOTTO. Any recommendations?
I know that if I use 5% BLOTTO that I can decrease this background signal
but I also decrease the antigen binding signal. I should add that the
starting antibody scFv does not demonstrate this non-specific binding,
so it appears to be something sticky within the library. In conclusion,
I do not find the methods in this system to be straightforward, but
instead rather empirical. Any help you can give would be greatly
If you can recommend any additional information sources besides
the currenly published literature I would greatly appreciate it as well.
I look forward to hearing from you.
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