Human Single Fold scFv Libraries I + J (Tomlinson I + J)
Over the past 10 years Greg Winter's lab at the MRC Laboratory
of Molecular Biology and the MRC Centre for
Protein Engineering (Cambridge, UK) has created a number of
artificial libraries of antibodies that can be used to derive
binders to almost any target molecule using phage display and
selection. These binders can be used for all the same applications
as conventional monoclonal antibodies (ELISA, Western blotting,
FACS, immunohistochemistry etc) but can be isolated in a fraction
of the time and without the need for animal immunisation. To date
these so called "naïve" or "single pot" phage-antibody libraries
have been used successfully in hundreds of molecular biology labs
world-wide to derive highly specific antibody reagents to a wide
range of different proteins, peptides or small molecule
compounds.
The latest libraries (Tomlinson I and J) that are being
distributed by Source BioScience LifeSciences each comprise over
100 million different scFv fragments cloned in an ampicillin
resistant phagemid vector and transformed into TG1 E. coli
cells (scFv fragments comprise a single polypeptide with the VH and
VL domains attached to one another by a flexible Glycine-Serine
linker). By carefully following the protocol provided in the
product data sheets below, large numbers of phagemids can be
produced and used to select specific binders to target molecules
that are attached to the surface of a tube or biotinylated and
captured by streptavidin coated beads (so called "panning"). After
each round of panning, the non-binders are washed away and the
phagemids bound to the target molecule/s are eluted and amplified
by infection into fresh TG1 cells. After producing new phagemids
from the previous round of panning, the process can be repeated.
Typically two or three rounds of panning are required to ensure
that more than half the different scFvs in the selected population
bind to the target molecule. The monoclonal scFvs can then be
screened for binding (using a simple ELISA based protocol) and then
used for further analysis of the target molecule. Since all the
functional scFvs in the Tomlinson I and J libraries bind Proteins A
and L, either of these secondary reagents can be used for
detection, purification or immobilisation. Alternatively, secondary
reagents that bind the attached myc or HIS6 tags can be used,
although in our experience it is better to use the Protein A or L
reagents.
For more details regarding this library you should read de Wildt et al, Nature Biotech vol 18,
2000 pp 989 - 993.
The protocol for the Human Single Fold scFv Libraries can be
found here.
The MRC Centre for Protein Engineering has a very helpful
compendium of questions and answers about antibody phage
display.
THIS LIBRARY IS PROVIDED AS A RESEARCH
TOOL AND IS NOT INTENDED TO BE USED
IN DRUG DISCOVERY AS THE BASIS FOR NOVEL THERAPEUTIC AGENTS.
THE MTA UNDER WHICH THIS PRODUCT IS RELEASED SETS OUT THE RIGHTS
THAT THE ORIGINATING ORGANISATION (MEDICAL RESEARCH COUNCIL) IS
ABLE TO GRANT. SHOULD YOU WISH TO COMMERCIALISE AN ANTIBODY
DEVELOPED FROM THIS LIBRARY, IT IS LIKELY THAT YOU WILL REQUIRE A
LICENSE FROM SEVERAL COMPANIES, AT LEAST ONE OF WHICH IS ONLY
AVAILABLE TO COMMERCIAL ENTITIES, NOT ACADEMIC ORGANISATIONS.
Useful links