We have been trying to construct ScFv antibodies using Pharmacia's
Recombinant Phage Antibody system. After hashing our way through various
technical problems, we finally got phage that gave a decent reaction by
ELISA. We tried to cut the ScFv DNA insert back out of the positive
clones, but didn't see the right size band on the gel. Finally tried PCR
using vector primers, and got a great product, but the wrong size. It
looks like we have cloned just the heavy or light chain variable region,
rather than the complete ScFv. This theoretically shouldn't be possible,
since only the complete ScFv has the NotI and SfiI sites for cloning. I
can see where maybe a small percentage of clones might end up with only
half the ScFv, but we have screened literally dozens without getting one
good one. This is very frustrating (and expensive!), and we have gotten
only one decent ScFv from three different hybridoma cell lines. Has anyone
else had this experience? I would also be happy to hear from anyone who
has successfully used this system to produce ScFv's. Did you follow the
protocol, or make your own modifications?
Thanks for your input.
Deborah Britt, Ph.D.
RI Hospital/Brown University