Open AccessIn vitro selection and affinity maturation of antibodies from a naive combinatorial immunoglobulin library.
Author(s) -
Hermann Gram,
Lori-Anne Marconi,
Carlos F. Barbas,
Thomas A. Collet,
Richard A. Lerner,
Angray S. Kang
Publication year - 1992
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.89.8.3576
Subject(s) - immunoglobulin light chain , antibody , hapten , phage display , peptide library , microbiology and biotechnology , conjugate , affinity maturation , monoclonal antibody , mutagenesis , bovine serum albumin , immunoglobulin heavy chain , biology , phagemid , complementarity determining region , chemistry , bacteriophage , biochemistry , immunology , escherichia coli , peptide sequence , mutation , gene , mathematical analysis , mathematics
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 mu and kappa 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, 10(4)-10(5) M-1) 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 mono-valent phage display vector pComb3 was performed by error-prone 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.