Pharmacia Recomb. phage Ab kit (ScFv)
RVDESHP0 at pwinet.upj.com
RVDESHP0 at pwinet.upj.com
Thu Oct 26 17:32:01 EST 1995
We had similar problems with the kit. We spent quite some time, money and
efforts figuring out what the problem could be. Pharmacia's tech-support
was of no significant help. Their answers were :
1."it is very TRICKY to get the assembly of VH and VL fragments done"
2."send us a photograph of your gel so that we can confirm that you are
using equal amounts of VH and VL PCR products in the assembly reaction"
3."different people have different judgments on how 50ng of VH or VL PCR
product DNA looks on a gel"
The tech-support could not offer one useful suggestion. On one fine day,
they suggested undertaking three rounds of biopanning and amplification of
ScFv phage library over antigen before testing in ELISA. We did that, but
without any success.
The problem lies in the degeneracy of the 5-prime VH and VL primers that
Pharmacia provides. We think that because of the degeneracy, these primers
may anneal to the target Ig mRNA (from hybridoma) even though there is some
mismatch. Since the hybridoma produces only one mRNA species for VH and VL,
the mismatched primers may yield a range of altered nucleotide sequences in
the PCR product, thereby giving several altered protein sequences of the
resultant ScFv. Furthermore, the mismatch annealing may also shift the
reading frame in the VH or VL fragment.
In any of the above conditions, the result would be identical : the
assembled ScFv will not bind the antigen. You will be able to amplify and
clone the DNA, but you may not produce the CORRECT PROTEIN.
In case of mRNA prepared from splenocytes of immunized mice, multiple mRNA
species for VH and VL will exist, and one of those WILL encode the V region
protein that binds the desired antigen.
In our case, we ended up amplifying the VH and VL regions from hybridomas
using Novagen's primers that bind outside the antibody framework regions,
and then sequencing those regions to find the EXACT sequence of the first
and the last framework regions. We then synthesized our own primers that
matched EXACTLY these sequences, re-amplified the VH, VL regions, and
assembled ScFv. We are testing these ScFv clones in ELISA at this time.
The conclusion from Pharmacia's kit is very simple : Don't take their word
(or technical excuses) for it. Change, adapt and succeed. Good luck.
Rajendra V. Deshpande, Ph.D.
Molecular Biology Research
The Upjohn Company
Kalamazoo, MI 49007
e-mail : rvdeshp0 at pwinet.upj.com
______________________________ Reply Separator _________________________________
Subject: Pharmacia Recomb. phage Ab kit (ScFv)
Author: debbritt at brownvm.brown.edu (Deb Britt) at INTERNET
Date: 10/26/95 1:03 PM
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 any input.
Deborah Britt, Ph.D.
RI Hospital/Brown University
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