Pharmacia Recombinant Phage Antibody kit?
H. David Fischer
davefischer at delphi.com
Thu Jun 16 18:31:58 EST 1994
Fourie Joubert <fourie at ccnet.up.ac.za> writes:
>Has anyone been using this kit? How suitable is it for creating libraries from
My lab has used both the old and new (E-tag) versions of the Pharmacia kit.
The results cloning from hybridomas are spotty. First, the primers, by
necessity, are degenerate and are suitable for only a limited set of Ig
gene families. The antibody from a given hybridoma might not amplify well
by PCR with them. Also, any given antibody binding site might not be
compatible with the linker used to make the single chain Fv construct; you
might get phage clones that don't bind or don't bind well. Also, we have
found some antibodies seem to be toxic for the E coli hosts. The expression
is a bit "leaky." If the antibody is "toxic," the cells select for non-
expressin mutants (usually frameshifts to generate stop codons, at least
in our hands). These issues are not specific to the kit----they are generic
problems to antibody phage display.
The results with libraries from spleen are much better---by definition, the
system selects for those antibodies that "work" and express well. In side-
by-side comparisons, though, we have still have more success getting mono-
clonals quicker and easier using standard hybridoma methods. The advantage to
the kit is that whatever antibodies you get are already cloned. That makes
any molecular analysis/manipulation easier. You will also likely get more
individual clones which may or may not be better for whatever binding activity
you are looking for: selection for high affinity still is a bit of an art.
Count on the whole business taking longer than the 8-11 days listed in
Pharmacia's literature, at least until you have experience. Finally, the
folks at Pharmacia's Milwaukee, WI labs can be very helpful and have technical
ties to the Cambridge Antibody Technology/MRC/Greg Winter labs.
More information about the Methods