78
Combinatorial Phage Antibody Libraries
formation, were transferred to nitrocel-
lulose Flters. Subsequently, the Flters
were probed with radiolabeled antigen,
and plaques formed by a phage encod-
ing antigen-binding ±ab were identiFed by
autoradiography.
The successes of the mouse system
were followed up by studies seeking to
apply the methodology for cloning hu-
man antibodies (±ig. 1). The Frst human
combinatorial ±abs were derived from a
library prepared using peripheral blood
lymphocytes (PBLs) of an individual re-
cently boosted with tetanus toxoid. No
speciFc ±abs, however, were obtained from
a similar but nonboosted tissue donor, de-
spite the high anti-tetanus toxoid serum
titer in this individual. This failure under-
scores an important point in constructing
human antibody libraries: in the absence
of recent boosting, PBLs are a poor source
of RNA from speciFc antibody-producing
Conversion to soluble
Fab expressing phagemid
Characterization of Fab
Discard
nonspecific
binders
Infect bacteria
Antigen
RNA
PBLs, bone marrow,
spleen
PCR,
library construction
Fig. 1
Strategy for cloning human
monoclonal Fab fragments from phage
display combinatorial libraries. RNA
prepared from antibody-producing
tissue sources (e.g. bone marrow and
peripheral blood) is reverse-transcribed,
andthelightcha
inandFdportionofthe
heavy chain are ampli±ed using PCR.
The ampli±ed genes are then cloned
sequentially into a phagemid vector and
‘‘rescued’’ to a phage display library in
which each phage expresses Fab on its
surface. The library is then ‘‘panned’’
over an immobilized antigen, and
speci±c phage Fabs are converted to a
soluble Fab-expressing system for
further characterization. [Adapted from
Burton, D.R. and Barbas, C.F. (1993)
Chem. lmmunol.
56
, 112–126.]
previous page 1398 Encyclopedia of Molecular Cell Biology and Molecular Medicine read online next page 1400 Encyclopedia of Molecular Cell Biology and Molecular Medicine read online Home Toggle text on/off