Antibody Molecules, Genetic Engineering of
339
B-cells harvested
from blood
V
H
genes
V
L
genes
PCR cloning of
V
H
and V
L
gene
repertoires
PCR
assembly and cloning
into a phage-display vector
Selection
1) Bind phage to antigen
2) Wash away nonbinding
phage
3) Elute binding phage and
amplify in
E. coli
4) Repeat 3 to 4 times
Round 1
Round 2
Round 3
Production
in
E. coli
culture
Screening
Desired scFv
Fig. 5
In vitro
human antibody production using phage libraries
(phage-display technology). Peripheral blood mononuclear cells are
harvested from a nonimmunized or immunized human donor, and the
heavy- and light-chain V-gene regions (V
L
and V
H
)areamp
liFedbyPCR
and assembled as a single-chain ±v region (sc±v). Note that the original
heavy- and light-chain pairings become scrambled during sc±v
assembly. The sc±v genes are cloned into Flamentous bacteriophages,
where the encoded sc±vs are displayed in a functional form on the phage
surface. Multiple rounds of selection with a solid-phase antigen allow
the isolation of even rare phages from the original library. The selected
sc±v can be expressed in
E. coli
(The illustration was adapted from
Powers D.B. and Marks J.D. Monovalent Phage Display of ±ab and sc±v
fusions. In
Antibody fusion proteins
. John Wiley & Son, Inc., New York,
1999).
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