Phage Display: A Powerful Technology for the Generation of High-Specificity Affinity Reagents from Alternative Immune Sources

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Abstract

Antibodies are critical reagents in many fundamental biochemical methods such as affinity chromatography, enzyme-linked immunosorbent assays (ELISA), flow cytometry, western blotting, immunoprecipitation, and immunohistochemistry techniques. As our understanding of the proteome becomes more complex, demand is rising for rapidly generated antibodies of higher specificity than ever before. It is therefore surprising that few investigators have moved beyond the classical methods of antibody production in their search for new reagents. Despite their long-standing efficacy, recombinant antibody generation technologies such as phage display are still largely the tools of biotechnology companies or research groups with a direct interest in protein engineering. In this chapter, we discuss the inherent limitations of classical polyclonal and monoclonal antibody generation and highlight an attractive alternative: generating high-specificity, high-affinity recombinant antibodies from alternative immune sources such as chickens, via phage display.

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Most cited references 60

Record: found
Abstract: found
Article: not found

Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli.

J Huston, D. Levinson, M Mudgett-Hunter … (1988)

A biosynthetic antibody binding site, which incorporated the variable domains of anti-digoxin monoclonal antibody 26-10 in a single polypeptide chain (Mr = 26,354), was produced in Escherichia coli by protein engineering. This variable region fragment (Fv) analogue comprised the 26-10 heavy- and light-chain variable regions (VH and VL) connected by a 15-amino acid linker to form a single-chain Fv (sFv). The sFv was designed as a prolyl-VH-(linker)-VL sequence of 248 amino acids. A 744-base-pair DNA sequence corresponding to this sFv protein was derived by using an E. coli codon preference, and the sFv gene was assembled starting from synthetic oligonucleotides. The sFv polypeptide was expressed as a fusion protein in E. coli, using a leader derived from the trp LE sequence. The sFv protein was obtained by acid cleavage of the unique Asp-Pro peptide bond engineered at the junction of leader and sFv in the fusion protein [(leader)-Asp-Pro-VH-(linker)-VL]. After isolation and renaturation, folded sFv displayed specificity for digoxin and related cardiac glycosides similar to that of natural 26-10 Fab fragments. Binding between affinity-purified sFv and digoxin exhibited an association constant [Ka = (3.2 +/- 0.9) x 10(7) M-1] that was about a factor of 6 smaller than that found for 26-10 Fab fragments [Ka = (1.9 +/- 0.2) x 10(8) M-1] under the same buffer conditions, consisting of 0.01 M sodium acetate, pH 5.5/0.25 M urea.

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Record: found
Abstract: found
Article: not found

Selection and identification of single domain antibody fragments from camel heavy-chain antibodies.

M Arbabi Ghahroudi, A Desmyter, L Wyns … (1997)

Functional heavy-chain gamma-immunoglobulins lacking light chains occur naturally in Camelidae. We now show the feasibility of immunising a dromedary, cloning the repertoire of the variable domains of its heavy-chain antibodies and panning, leading to the successful identification of minimum sized antigen binders. The recombinant binders are expressed well in E. coli, extremely stable, highly soluble, and react specifically and with high affinity to the antigens. This approach can be viewed as a general route to obtain small binders with favourable characteristics and valuable perspectives as modular building blocks to manufacture multispecific or multifunctional chimaeric proteins.

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Record: found
Abstract: found
Article: not found

Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides.

J Wölle, Sarah Fischer, Andreas Plückthun … (2000)

By analyzing the human antibody repertoire in terms of structure, amino acid sequence diversity and germline usage, we found that seven V(H) and seven V(L) (four Vkappa and three Vlambda) germline families cover more than 95 % of the human antibody diversity used. A consensus sequence was derived for each family and optimized for expression in Escherichia coli. In order to make all six complementarity determining regions (CDRs) accessible for diversification, the synthetic genes were designed to be modular and mutually compatible by introducing unique restriction endonuclease sites flanking the CDRs. Molecular modeling verified that all canonical classes were present. We could show that all master genes are expressed as soluble proteins in the periplasm of E. coli. A first set of antibody phage display libraries totalling 2x10(9) members was created after cloning the genes in all 49 combinations into a phagemid vector, itself devoid of the restriction sites in question. Diversity was created by replacing the V(H) and V(L) CDR3 regions of the master genes by CDR3 library cassettes, generated from mixed trinucleotides and biased towards natural human antibody CDR3 sequences. The sequencing of 257 members of the unselected libraries indicated that the frequency of correct and thus potentially functional sequences was 61 %. Selection experiments against many antigens yielded a diverse set of binders with high affinities. Due to the modular design of all master genes, either single binders or even pools of binders can now be rapidly optimized without knowledge of the particular sequence, using pre-built CDR cassette libraries. The small number of 49 master genes will allow future improvements to be incorporated quickly, and the separation of the frameworks may help in analyzing why nature has evolved these distinct subfamilies of antibody germline genes. Copyright 2000 Academic Press.

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Author and article information

Contributors

Dermot Walls: +35335317005600 , +35335317005412 , dermot.walls@dcu.ie

Sinéad T. Loughran: +353353+353-1-700-8683 , +353353+353-1-700-5412 , sinead.loughran@dkit.ie

Orla Cunningham: orla.cunningham@pfizer.com

Journal

Journal ID (publisher-id): 978-1-4939-6412-3

Journal ID (doi): 10.1007/978-1-4939-6412-3

Journal ID (nlm-ta): Protein Chromatography

Title: Protein Chromatography

Subtitle: Methods and Protocols

ISBN (Print): 978-1-4939-6410-9

ISBN (Electronic): 978-1-4939-6412-3

Publication date (Electronic): 23 May 2016

Publication date Collection: 2017

Volume: 1485

Pages: 85-99

Affiliations

[2 ]GRID grid.15596.3e, ISNI 0000000102380260, School of Biotechnology, , Dublin City University, ; Dublin, Ireland

[3 ]GRID grid.418613.9, ISNI 0000000417566094, Department of Applied Sciences, , Dundalk Institute of Technology, ; Dublin, Ireland

[4 ]GRID grid.482367.c, ISNI 0000 0004 0401 4901, Global Biotherapeutics Technologies, , Pfizer, ; Grange Castle Business Park, Clondalkin, Dublin 22, Ireland

[5 ]GRID grid.410513.2, ISNI 0000000088007493, Pfizer Inc., ; 610 Main St., Cambridge, MA 02139 USA

Article

Publisher ID: 6

DOI: 10.1007/978-1-4939-6412-3_6

PMC ID: 7153406

PubMed ID: 27730550

SO-VID: 03f8e3c4-99fb-4d4e-98cf-ca947b059c21

License:

This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.