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EMBO J. 1994 Jul 15;13(14):3245-60.

Isolation of high affinity human antibodies directly from large synthetic repertoires.

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1
MRC Centre for Protein Engineering, Cambridge, UK.

Abstract

Antibody fragments of moderate affinity (approximately microM) can be isolated from repertoires of approximately 10(8) immunoglobulin genes by phage display and rounds of selection with antigen, and the affinities improved by further rounds of mutation and selection. Here, as an alternative strategy, we attempted to isolate high affinity human antibodies directly from large repertoires. We first created highly diverse repertoires of heavy and light chains entirely in vitro from a bank of human V gene segments and then, by recombination of the repertoires in bacteria, generated a large (close to 6.5 x 10(10)) synthetic repertoire of Fab fragments displayed on filamentous phage. From this repertoire we isolated Fab fragments which bound to a range of different antigens and haptens, and with affinities comparable with those of antibodies from a secondary immune response in mice (up to 4 nM). Although the VH-26 (DP-47) segment was the most commonly used segment in both artificial and natural repertoires, there were also major differences in the pattern of segment usage. Such comparisons may help dissect the contributions of biological mechanisms and structural features governing V gene usage in vivo.

PMID:
8045255
PMCID:
PMC395221
[Indexed for MEDLINE]
Free PMC Article
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