The Center will perform 2 to 4 rounds of "panning"
with the Mehta libraries against a purified polypeptide or protein targets of
interest. We will screen 200 clones by ELISA and up to 100 positive clones will
be further examined by DNA sequencing to identify "unique" clones.
The Center will INITALLY supply investigators with glycerol stocks of transformed
E. coli and simple protocols for the purification of antibodies from
E. coli periplasmic preparations. NFCR Project Directors can choose
any of the options listed below.
Option 1. NFCR Project Directors
can obtain a glycerol stock of transformed E. coli (TG1 strain) that
can be used to rescue the human sFv-phage which can be directly detected with
commercial anti-M13 antisera and/or anti-c-myc Mab.
Option 2. NFCR Project Directors
can obtain a glycerol stock of transformed E. coli (HB2151 strain)
that can be used to make periplasmic preparations containing soluble c-myc tagged
sFv antibodies with yields of circa 50 ug to 500 ug/liter.
Option 3. NFCR Project Directors
can obtain a pUC19-based bacterial expression vector for rapid cloning, expression
and purification of c-myc-His6 tagged sFv antibodies from periplasmic
preparations with yields of 100 ug to 1 mg/liter.
Option 4. This option is
only available after the Project Director has performed primary functional screens
and has determined that a specific subset of sFv proteins should be evaluated
in more detail. Upon special arrangement, CTAE will produce highly purified
sFvs from 1-liter flasks of E. coli cultures that can be used in further
As expansion of the CTAE activities occurs, future output from the Center
will be extended to the following areas:
- Biacore to measure antibody affinities, kon and koff rates
- Eukaryotic expression and purification of human sFv-IgG fusion proteins.
- Construction and expression of full human monoclonal antibodies.
- Intracellular antibody "Intrabody" expression vectors for gene
"knock-out" and gene therapy experiments.
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