7 research outputs found
On the use of combinatorial antibody libraries to clone the "fossil record" of an individual's immune response
For about the last century the record of
immunological events could only be obtained from serum
proteins. We suggest that in the future this will change and the
far more detailed nucleic acid record will provide new insights
into immunological processes as well as selective access to the
complete repertoire
Linkage of recognition and replication functions by assembling combinatorial antibody Fab libraries along phage surfaces
We describe a method based on a phagemid
vector with helper phage rescue for the construction and rapid
analysis of combinatorial antibody Fab libraries. This approach
should allow the generation and selection of many
monoclonal antibodies. Antibody genes are expressed in concert
with phage morphogenesis, thereby allowing incorporation
of functional Fab molecules along the surface of ifiamentous
phage. The power of the method depends upon the
linkage of recognition and replication functions and is not
limited to antibody molecules
In vitro selection and affinity maturation of antibodies from a naive combinatorial immunoglobulin library
We have used a combinatorial immunoglobulin
library approach to obtain monoclonal antibodies from
nonimmune adult mice, thereby establishing the principles of
(i) accessing naive combinatorial antibody libraries for predetermined
specificities and (ii) increasing the affinity of the
selected antibody binding sites by random mutagenesis. A
combinatorial Fab library expressing immunoglobulin Ip and it
light-chain fragments on the surface of filamentous phage was
prepared from bone marrow of nonimmunized, adult BALB/c
mice with the multivalent display vector pComb8. Phage
displaying low affinity Fabs (binding constants, 104-1O M-')
binding to a progesterone-bovine serum albumin conjugate
were isolated from the library. Random mutagenesis of the
heavy- and light-chain variable regions expressed in the monovalent
phage display vector pComb3 was performed by errorprone
PCR, and subsequently clones with improved affinity for
the hapten conjugate were selected. We demonstrate that
antibodies with desirable characteristics from a nonimmune
source may be selected and affinity maturation may be achieved
by using the twin vectors pComb8 and pComb3, thus opening
the route to obtaining specific antibodies from a generic library
and bypassing immunization