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Phage Display

A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Biochemistry".

Deadline for manuscript submissions: closed (29 February 2012) | Viewed by 60365

Special Issue Editors

Australian Institute for Bioengineering and Nanotechnology (AIBN), Corner College and Cooper Rds (Bldg 75), The University of Queensland, Brisbane Qld 4072, Australia
Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, Brisbane, QLD 4072, Australia

Special Issue Information

Dear Colleagues,

Antibodies have proven to be one of the most important classes of biological molecules with widespread use as research, diagnostic and therapeutic reagents, due to their unique and high affinity binding to a diverse range of targets. In vitro display technology, such as the display of antibody fragments on the surface of M13, fd and f1 filamentous bacteriophage (phage display), offers a highly powerful method capable of generating antibodies to any desired target with specificity and affinity that is not achievable using established hybridoma approaches [1].

The success or failure of in vitro display is dependent on a number of variables including;

  • Size and diversity of immunoglobulin gene library
  • Quality and purity of target antigen
  • Complexity of target
  • Biopanning strategies

Immunoglobulin gene libraries can be composed of natural antibody sequences isolated from either naïve or immunised antibody-producing cells, or can be synthesised to contain random complementarity-determining sequences, allowing isolation of non-naturally occurring binders. Antigens for panning are commonly purified native or recombinant proteins, or whole cells or viruses, but may also be non-protein samples such as lipids or carbohydrates.

The isolation of antibodies against a given target antigen is relatively straight forward, and three to four iterations (rounds) of biopanning generally result in the isolation of one or more antibodies to different epitopes located on the target. However there are more complex scenarios for isolation of antibodies against targets, which require designing specific strategies for biopanning, often incorporating combinations of negative selection steps, to deplete the repertoire of irrelevant antibodies. Such examples include isolation of antibodies against viral antigens that are able to discriminate between closely related subtypes [2], or panning against whole cells where the desired target is only a fraction of the available epitopes present on the cell.

Phage display is ideal for such complex scenarios as experimental conditions can easily be altered such as stringency of selection or negative depletion. Additionally, isolated antibodies can easily be mutagenised for affinity maturation.

Prof. Dr. Stephen Mahler
Dr. Martina Jones
Guest Editors

References

1. Smith, G.P.; Petrenko, V.A. Phage Display. Chem. Rev. 1997, 97, 391–410.
2. Bradbury, A.R.M.; Sidhu, S.; Dubel, S.; McCafferty, J. Beyond natural antibodies: the power of in vitro display technologies. Nat. Biotechnol. 2011, 29, 245–254

Keywords

  • antibodies
  • biopanning
  • phage display
  • subtractive selection
  • whole cell panning

Published Papers (7 papers)

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Research

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529 KiB  
Article
Generation of a Highly Reactive Chicken-Derived Single-Chain Variable Fragment against Fusarium verticillioides by Phage Display
by Zu-Quan Hu, Jin-Long Liu, He-Ping Li, Shu Xing, Sheng Xue, Jing-Bo Zhang, Jian-Hua Wang, Greta Nölke and Yu-Cai Liao
Int. J. Mol. Sci. 2012, 13(6), 7038-7056; https://doi.org/10.3390/ijms13067038 - 07 Jun 2012
Cited by 16 | Viewed by 7920
Abstract
Fusarium verticillioides is the primary causal agent of Fusarium ear and kernel rot in maize, producing fumonisin mycotoxins that are toxic to humans and domestic animals. Rapid detection and monitoring of fumonisin-producing fungi are pivotally important for the prevention of mycotoxins from entering [...] Read more.
Fusarium verticillioides is the primary causal agent of Fusarium ear and kernel rot in maize, producing fumonisin mycotoxins that are toxic to humans and domestic animals. Rapid detection and monitoring of fumonisin-producing fungi are pivotally important for the prevention of mycotoxins from entering into food/feed products. Chicken-derived single-chain variable fragments (scFvs) against cell wall-bound proteins from F. verticillioides were isolated from an immunocompetent phage display library. Comparative phage enzyme-linked immunosorbant assays (ELISAs) and sequencing analyses identified four different scFv antibodies with high sensitivity. Soluble antibody ELISAs identified two highly sensitive scFv antibodies, FvCA3 and FvCA4, with the latter being slightly more sensitive. Three-dimensional modeling revealed that the FvCA4 may hold a better overall structure with CDRH3, CDRL1 and CDRL3 centered in the core region of antibody surface compared with that of other scFvs. Immunofluorescence labeling revealed that the binding of FvCA4 antibody was localized to the cell walls of conidiospores and hyphae of F. verticillioides, confirming the specificity of this antibody for a surface target. This scFv antibody was able to detect the fungal mycelium as low as 10−2 μg/mL and contaminating mycelium at a quantity of 10−2 mg/g maize. This is the first report that scFv antibodies derived from phage display have a wide application for rapid and accurate detection and monitoring of fumonisin-producing pathogens in agricultural samples. Full article
(This article belongs to the Special Issue Phage Display)
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1234 KiB  
Article
Identification of Late Embryogenesis Abundant (LEA) Protein Putative Interactors Using Phage Display
by Rekha Kushwaha, Taylor D. Lloyd, Kim R. Schäfermeyer, Santosh Kumar and Allan Bruce Downie
Int. J. Mol. Sci. 2012, 13(6), 6582-6603; https://doi.org/10.3390/ijms13066582 - 29 May 2012
Cited by 12 | Viewed by 7650
Abstract
Arabidopsis thaliana seeds without functional SEED MATURATION PROTEIN1 (SMP1), a boiling soluble protein predicted to be of intrinsic disorder, presumed to be a LATE EMBRYOGENESIS ABUNDANT (LEA) family protein based on sequence homology, do not enter secondary dormancy after 3 days at 40 [...] Read more.
Arabidopsis thaliana seeds without functional SEED MATURATION PROTEIN1 (SMP1), a boiling soluble protein predicted to be of intrinsic disorder, presumed to be a LATE EMBRYOGENESIS ABUNDANT (LEA) family protein based on sequence homology, do not enter secondary dormancy after 3 days at 40 °C. We hypothesized that SMP1 may protect a heat labile protein involved in the promotion of secondary dormancy. Recombinant SMP1 and GmPM28, its soybean (Glycine max), LEA4 homologue, protected the labile GLUCOSE-6-PHOSPHATE DEHYROGENASE enzyme from heat stress, as did a known protectant, Bovine Serum Albumin, whether the LEA protein was in solution or attached to the bottom of microtiter plates. Maintenance of a biological function for both recombinant LEA proteins when immobilized encouraged a biopanning approach to screen for potential protein interactors. Phage display with two Arabidopsis seed, T7 phage, cDNA libraries, normalized for transcripts present in the mature, dehydrated, 12-, 24-, or 36-h imbibed seeds, were used in biopans against recombinant SMP1 and GmPM28. Phage titer increased considerably over four rounds of biopanning for both LEA proteins, but not for BSA, at both 25 and at 41 °C, regardless of the library used. The prevalence of multiple, independent clones encoding portions of specific proteins repeatedly retrieved from different libraries, temperatures and baits, provides evidence suggesting these LEA proteins are discriminating which proteins they protect, a novel finding. The identification of putative LEA-interacting proteins provides targets for reverse genetic approaches to further dissect the induction of secondary dormancy in seeds in response to heat stress. Full article
(This article belongs to the Special Issue Phage Display)
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261 KiB  
Article
Generation and Characterization of a Novel Recombinant Antibody Against 15-Ketocholestane Isolated by Phage-Display
by Md. Omedul Islam, Yan Ting Lim, Conrad En Zuo Chan, Amaury Cazenave-Gassiot, J. Ludovic Croxford, Markus R. Wenk, Paul A. Macary and Brendon J. Hanson
Int. J. Mol. Sci. 2012, 13(4), 4937-4948; https://doi.org/10.3390/ijms13044937 - 19 Apr 2012
Cited by 5 | Viewed by 6702
Abstract
The employment of monoclonal antibodies (Mabs) to identify disease-associated biomarkers in clinical samples represents the underlying principle for many diagnostic tests. To date, these have been principally developed for protein targets with few reported applications for lipids due to their hydrophobicity and poor [...] Read more.
The employment of monoclonal antibodies (Mabs) to identify disease-associated biomarkers in clinical samples represents the underlying principle for many diagnostic tests. To date, these have been principally developed for protein targets with few reported applications for lipids due to their hydrophobicity and poor immunogenicity. Oxysterols represent a family of lipids implicated in diverse human diseases where Mab-based detection assays could have a profound effect on their utility as clinical biomarkers. These are usually identified in patients’ samples by mass- spectrometry based approaches. Here, we describe an antibody phage-library based screening methodology for generating a recombinant monoclonal antibody (RAb) targeting the oxysterol-15-ketocholestane (15-KA), a lipid implicated in multiple sclerosis and Autoimmune Encephalomyelitis (EAE). The antibody is highly specific for 15-KA and shows little or no binding activity for other closely related oxysterols. We employ RAb2E9 to address the controversy over whether 15-KA is a true biomarker for MS/EAE and show that 15-KA is undetectable in serum taken from mice with EAE using antibody based detection methodologies; a finding confirmed by mass-spectrometry analysis. This study demonstrates the technical feasibility of using phage display to isolate highly specific antibodies against poorly immunogenic, small molecule lipids. Full article
(This article belongs to the Special Issue Phage Display)
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629 KiB  
Article
Phage Display Approaches for the Isolation of Monoclonal Antibodies Against Dengue Virus Envelope Domain III from Human and Mouse Derived Libraries
by Nicole J. Moreland, Patricia Susanto, Elfin Lim, Moon Y. F. Tay, Ravikumar Rajamanonmani, Brendon J. Hanson and Subhash G. Vasudevan
Int. J. Mol. Sci. 2012, 13(3), 2618-2635; https://doi.org/10.3390/ijms13032618 - 27 Feb 2012
Cited by 15 | Viewed by 7663
Abstract
Domain III of the dengue virus envelope protein (EDIII, aa295-395) has an immunoglobulin fold and is the proposed receptor-binding domain of the virus. Previous studies have shown that monoclonal antibodies against EDIII can be neutralizing and have therapeutic potential. Here, cloned Fab-phage libraries [...] Read more.
Domain III of the dengue virus envelope protein (EDIII, aa295-395) has an immunoglobulin fold and is the proposed receptor-binding domain of the virus. Previous studies have shown that monoclonal antibodies against EDIII can be neutralizing and have therapeutic potential. Here, cloned Fab-phage libraries of human and mouse origin were screened for DENV specific antibodies. Firstly, bacterially expressed EDIII or whole virus particles were used as bait in biopanning against a large naïve human Fab-phage library ( > 10 billion independent clones). Multiple panning strategies were employed, and in excess of 1000 clones were screened, but all of the antibodies identified bound the envelope in regions outside EDIII suggesting EDIII antibodies are virtually absent from the naïve human repertoire. Next, a chimeric Fab-phage library was constructed from a panel of EDIII specific mouse hybridomas by pooling the VH and VL chain sequences from the hybridomas and cloning these into the pComb3X phagemid vector with human CH and CL encoding sequences. Biopanning against EDIII identified a unique antibody (C9) that cross-reacts with EDIII from DENV1-3 and, in the IgG format, binds and neutralizes DENV2 in cell-based assays. Sequence analysis and saturation mutagenesis of complementary determining regions (CDR) in the C9 light chain suggest an antigen recognition model in which the LCDR3 is a key determinant of EDIII specificity, while modifications in LCDR1 and LCDR2 affect DENV serotype cross-reactivity. Overall, this study supports the current prevailing opinion that neutralizing anti-EDIII monoclonal antibodies can be readily generated in murine systems, but in humans the anti-DENV immune response is directed away from domain III. Full article
(This article belongs to the Special Issue Phage Display)
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Review

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390 KiB  
Review
Phage Displayed Peptides/Antibodies Recognizing Growth Factors and Their Tyrosine Kinase Receptors as Tools for Anti-Cancer Therapeutics
by Roberto Ronca, Patrizia Benzoni, Angela De Luca, Elisabetta Crescini and Patrizia Dell’Era
Int. J. Mol. Sci. 2012, 13(4), 5254-5277; https://doi.org/10.3390/ijms13045254 - 24 Apr 2012
Cited by 6 | Viewed by 7873
Abstract
The basic idea of displaying peptides on a phage, introduced by George P. Smith in 1985, was greatly developed and improved by McCafferty and colleagues at the MRC Laboratory of Molecular Biology and, later, by Barbas and colleagues at the Scripps Research Institute. [...] Read more.
The basic idea of displaying peptides on a phage, introduced by George P. Smith in 1985, was greatly developed and improved by McCafferty and colleagues at the MRC Laboratory of Molecular Biology and, later, by Barbas and colleagues at the Scripps Research Institute. Their approach was dedicated to building a system for the production of antibodies, similar to a naïve B cell repertoire, in order to by-pass the standard hybridoma technology that requires animal immunization. Both groups merged the phage display technology with an antibody library to obtain a huge number of phage variants, each of them carrying a specific antibody ready to bind its target molecule, allowing, later on, rare phage (one in a million) to be isolated by affinity chromatography. Here, we will briefly review the basis of the technology and the therapeutic application of phage-derived bioactive molecules when addressed against key players in tumor development and progression: growth factors and their tyrosine kinase receptors. Full article
(This article belongs to the Special Issue Phage Display)
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215 KiB  
Review
Filamentous Bacteriophage Fd as an Antigen Delivery System in Vaccination
by Antonella Prisco and Piergiuseppe De Berardinis
Int. J. Mol. Sci. 2012, 13(4), 5179-5194; https://doi.org/10.3390/ijms13045179 - 24 Apr 2012
Cited by 48 | Viewed by 10733
Abstract
Peptides displayed on the surface of filamentous bacteriophage fd are able to induce humoral as well as cell-mediated immune responses, which makes phage particles an attractive antigen delivery system to design new vaccines. The immune response induced by phage-displayed peptides can be enhanced [...] Read more.
Peptides displayed on the surface of filamentous bacteriophage fd are able to induce humoral as well as cell-mediated immune responses, which makes phage particles an attractive antigen delivery system to design new vaccines. The immune response induced by phage-displayed peptides can be enhanced by targeting phage particles to the professional antigen presenting cells, utilizing a single-chain antibody fragment that binds dendritic cell receptor DEC-205. Here, we review recent advances in the use of filamentous phage fd as a platform for peptide vaccines, with a special focus on the use of phage fd as an antigen delivery platform for peptide vaccines in Alzheimer’s Disease and cancer. Full article
(This article belongs to the Special Issue Phage Display)
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848 KiB  
Review
Phages and HIV-1: From Display to Interplay
by Sylvie Delhalle, Jean-Claude Schmit and Andy Chevigné
Int. J. Mol. Sci. 2012, 13(4), 4727-4794; https://doi.org/10.3390/ijms13044727 - 13 Apr 2012
Cited by 18 | Viewed by 11203
Abstract
The complex hide-and-seek game between HIV-1 and the host immune system has impaired the development of an efficient vaccine. In addition, the high variability of the virus impedes the long-term control of viral replication by small antiviral drugs. For more than 20 years, [...] Read more.
The complex hide-and-seek game between HIV-1 and the host immune system has impaired the development of an efficient vaccine. In addition, the high variability of the virus impedes the long-term control of viral replication by small antiviral drugs. For more than 20 years, phage display technology has been intensively used in the field of HIV-1 to explore the epitope landscape recognized by monoclonal and polyclonal HIV-1-specific antibodies, thereby providing precious data about immunodominant and neutralizing epitopes. In parallel, biopanning experiments with various combinatorial or antibody fragment libraries were conducted on viral targets as well as host receptors to identify HIV-1 inhibitors. Besides these applications, phage display technology has been applied to characterize the enzymatic specificity of the HIV-1 protease. Phage particles also represent valuable alternative carriers displaying various HIV-1 antigens to the immune system and eliciting antiviral responses. This review presents and summarizes the different studies conducted with regard to the nature of phage libraries, target display mode and biopanning procedures. Full article
(This article belongs to the Special Issue Phage Display)
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