Combinatorial Phage Antibody Libraries
85
homogeneity from bacterial supernatants
in a single step by passage over an anti-Fab
af±nity column.
For
many
applications
of
antibod-
ies, whole immunoglobin molecules are
preferred. To this end, a number of vec-
tor systems have been assembled, which
facilitate rapid subcloning from phage
display vectors to eukaryotic expression
systems. This presents the opportunity
for the investigator to create whole an-
tibodies of any isotype from a phage-
selected Fab by joining variable region
sequences to the appropriate constant
regions contained within the eukaryotic
vector.
4
Applications of Combinatorial Antibody
Libraries
4.1
Antibodies from Immune Donors
Recombinant
antibodies
isolated
from
pComb3-based
combinatorial
libraries
Fig. 4
Panning of the combinatorial
phage display library. Phages are
incubated with immobilized antigen (a),
and unbound phages are removed by
washing (b). The remaining phages are
eluted in a low pH solution or in the
presence of excess soluble antigen (c,
d). Eluted phages are then reinfected
into bacteria, reampliFed, and reapplied
to the antigen. Three or four repetitions
of steps (a) to (d) result in a progressive
enrichment for antigen-speciFc
phage-±ab. Phages from (d) are
converted to the phagemid form of the
vector, the DNA prepared, and the
gene3
sequence excised. Religation then yields
a reconstructed phagemid, which may
be used to transform bacteria for the
production of soluble ±ab (e). [Adapted
from Burton, D.R. (1992)
Hosp. Prac.
27
,
67–74.]
Phage
Antibody
Fab
Phage display library
Antigen
(a)
(b)
(c)
(d)
(e)
Reconstruct phagemid
Fab phage display
Soluble expression
Isolate
specific
phage-Fab
Soluble
Fab
Elute at low pH
were ±rst prepared from antigenically
boosted mice and humans. Speci±c Fabs
have also been generated from humans
who have not been actively boosted but
whohavea
tsomet
imehadcon
tac
tw
i
th
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