A human scFv antibody generation pipeline for proteome research

The functional decryption of the human proteome is the challenge which follows the sequencing of the human genome. Specific binders to every human protein are key reagents for this purpose. In vitro antibody selection using phage display offers one possible solution that can meet the demand for 25,0...

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Bibliographic Details
Published in:Journal of biotechnology 2011-04, Vol.152 (4), p.159-170
Main Authors: Hust, Michael, Meyer, Torsten, Voedisch, Bernd, Rülker, Torsten, Thie, Holger, El-Ghezal, Aymen, Kirsch, Martina Inga, Schütte, Mark, Helmsing, Saskia, Meier, Doris, Schirrmann, Thomas, Dübel, Stefan
Format: Article
Language:English
Subjects:
antibodies
Antibody engineering
bacterial proteins
bacteriophages
biotechnology
Biotechnology - methods
biotinylation
CD71
Cell Line
DNA libraries
DNA Primers - genetics
Escherichia coli
fungi
Gene Library
genes
Genetic Vectors - genetics
haptens
Human proteome research
Humans
Immunoglobulin
mAb, Monoclonal antibody
packaging
Panning
Phage display
proteome
Proteomics - methods
scFv
scFv-Fc
Single-Chain Antibodies - biosynthesis
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Summary:The functional decryption of the human proteome is the challenge which follows the sequencing of the human genome. Specific binders to every human protein are key reagents for this purpose. In vitro antibody selection using phage display offers one possible solution that can meet the demand for 25,000 or more antibodies, but needs substantial standardisation and minimalisation. To evaluate this potential, three human, naive antibody gene libraries (HAL4/7/8) were constructed and a standardised antibody selection pipeline was set up. The quality of the libraries and the selection pipeline was validated with 110 antigens, including human, other mammalian, fungal or bacterial proteins, viruses or haptens. Furthermore, the abundance of VH, kappa and lambda subfamilies during library cloning and the E. coli based phage display system on library packaging and the selection of scFvs was evaluated from the analysis of 435 individual antibodies, resulting in the first comprehensive comparison of V gene subfamily use for all steps of an antibody phage display pipeline. Further, a compatible cassette vector set for E. coli and mammalian expression of antibody fragments is described, allowing in vivo biotinylation, enzyme fusion and Fc fusion.
ISSN:0168-1656
1873-4863
DOI:10.1016/j.jbiotec.2010.09.945