key: cord-257465-9yrf7ofy
authors: Finlay, William J. J.; Bloom, Laird; Grant, Joanne; Franklin, Edward; Shúilleabháin, Deirdre Ní; Cunningham, Orla
title: Phage Display: A Powerful Technology for the Generation of High-Specificity Affinity Reagents from Alternative Immune Sources
date: 2016-05-23
journal: Protein Chromatography
DOI: 10.1007/978-1-4939-6412-3_6
sha: 
doc_id: 257465
cord_uid: 9yrf7ofy

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.

The rapid expansion of the genomics, proteomics , and biotechnology fi elds has led to a growing demand for affi nity reagents that can specifi cally recognize proteins, peptides, carbohydrates, and haptens . Affi nity reagents of high specifi city are routinely required for diverse protein drug targets, members of newly discovered biochemical pathways, posttranslationally modifi ed proteins, protein cleavage products, and even small molecules such as drugs of abuse and toxins. Individual biomedical researchers will often need to monitor, quantify, and purify proteins of interest via affi nity chromatography , but there may not be any commercially available antibody reagents to allow them to do so [ 1 ] . Indeed, even in situations where there are commercially available antibodies, these reagents are often expensive, poorly characterized, and/or simply not appropriate for demanding applications. Compounding this problem, the technical diffi culty of monoclonal antibody generation by the untrained researcher and the high cost (~$15,000) of a commercial monoclonal antibody generation program leads many researchers to the default solution of producing polyclonal hyperimmune sera in hosts such as rabbits. The net result of this is that researchers often settle for reagents that lack the necessary specifi city to perform the applications for which they were intended.

In this review, we will outline the limitations of classical antibody generation technologies and illustrate an attractive alternative: the use of phage display libraries of recombinant antibodies built on immunoglobulin repertoires from nonmammalian animals. In particular, we will highlight the advantages of libraries derived from the domestic chicken Gallus gallus , which offers a relatively inexpensive and technically accessible route to high-quality monoclonal reagents [ 2 ] . If, like many people, you have purchased (or paid to generate) a costly and "specifi c" antibody, but subsequently found that it is actually polyreactive and of dubious quality, phage display from immunized chickens may offer an attractive alternative.

Hyper-immune sera from rabbits, sheep, or other mammals may be produced in large quantities, but they do not offer the consistency of monoclonal antibodies and need to be regularly replenished and recharacterized. Serum antibodies are also polyclonal and frequently polyspecifi c, even when purifi ed over an antigen column, rendering them suboptimal for the specifi c recognition of a single component in a complex matrix. One illuminating study has demonstrated that when used to probe a comprehensive yeast proteome chip, unpurifi ed polyclonal antibody preparations could recognize up to 1770 different proteins, with some monoclonal antibodies and antigen column-purifi ed polyclonal antibodies also recognizing multiple proteins (related and unrelated) [ 3 ] .

The arrival of monoclonal antibody technology [ 4 ] was a major step forward in generating high-specifi city reagents, but the reliance on the murine immunoglobulin system frequently leads to a number of practical diffi culties: (1) Monoclonal antibodies are raised on the basis of an ineffi cient fusion of splenic B-cells to an immortalized mouse myeloma line, followed by limiting dilution of the cell population. Target-specifi c antibodies are randomly identifi ed, often by a simple direct ELISA , where few preconditions can be set to determine which antibodies are identifi ed and one must "take what one can get" during the screening process. (2) It is often desirable to have multiple monoclonal antibodies with specifi city for different epitopes on the same target molecule, but the diffi culty in sequencing monoclonals does not allow the rapid identifi cation of unique clones early in the screening process. (3) Humans and rodents are relatively closely related phylogenetically. Many proteins of interest are highly

conserved among mammals and this can frequently lead to thymic tolerance, restricting the antibody response after immunization. (4) When an immune response to a human protein is raised in mice, the large regions of sequence similarity between murine and human proteins may lead to a restricted number of immunogenic epitopes. (5) To generate antibodies that cross-react with orthologues from multiple species of mammal is particularly tricky, as the common epitopes among mammals are the very ones that are unlikely to provoke a strong immunoglobulin response in the mouse. (6) Tolerance issues can become even harder to circumvent when the protein of interest is from a mouse or rat. Creating "knockout" mice, in which the endogenous copy of the gene for the target protein has been disabled, can often break tolerance, but this is a highly laborious and time-consuming process that few laboratories have the resources to undertake. These factors all hinder the generation of high-quality antibody reagents and thereby limit one's experimental options when developing antibodies for purifying or tracking novel proteins.

To bypass the limitations in polyclonal and monoclonal antibody generation, several groups have turned to in vitro display technologies such as phage display, ribosome display, or yeast display libraries to generate recombinant antibodies. The more recent technologies of yeast and ribosome display are becoming highly established, but phage display is currently the most robust, well characterized, and reliable of these methods. Antibodies derived from these technologies are cloned in microbial hosts and are therefore monoclonal from the start, with their production being easily scaled up. Critically, selection and screening efforts can be directed toward specifi c epitopes or species cross-reactivity and away from polyspecifi c binding. Phage display allows a researcher to do in a single tube what would be unfathomable in traditional hybridoma work: interrogate libraries of millions to tens of billions of antibodies on the basis of their binding specifi cally to a target of interest.

All of this is possible due to the ingenious concept of "genotypeto-phenotype linkage," which is exemplifi ed by phage display. Phage display was originally described as a rapid method for cloning gene fragments that encode a specifi c protein [ 5 ] . By cloning gene fragments into the genome of a fi lamentous E. coli bacteriophage, Smith et al. were able to generate libraries of gene fusions with the key phage coat protein p3. After transformation into E. coli , the viral replication system packaged the genome (and therefore the cloned gene) into a highly stable complex carrying the gene product on the tip of the phage particle, as a fusion protein with p3 . By subsequently selecting expressed virions on an immobilized antibody with specifi city for a known protein that had been cloned in the phage genome, they were able to show 1000-fold enrichment of the gene product. This set of experiments showed that "genotypeto-phenotype linkage" could be achieved and thereby defi ned the basis of all display technologies developed since.

The subsequent development of a method for the effective cloning of antibody V-gene sequences via PCR allowed the capture of antibody sequences in a recombinant form [ 6 ] , removing the need to immortalize B-cells via hybridoma fusion as required in traditional monoclonal antibody generation. This discovery was combined with the phage display process to make an effi cient method of isolating antibodies and their corresponding gene sequences simultaneously [ 7 ] . In the antibody phage display process, libraries of diverse V-gene sequences are cloned into an appropriate expression vector in E. coli , creating an in-frame fusion with the p3 protein or, as favored in most recently described libraries, a truncated form of p3.

The phage display of protein libraries was originally performed using "phage" vectors (i.e., built upon the phage genome itself), but due to practical diffi culties in handling these libraries, more recent phage methods have mostly used so-called phagemid expression vectors. In this case, the DNA backbone is a stable, small plasmid such as pUC, and the p3 gene plus f1 phage packaging origin are the only phage-derived DNA sequences [ 8 ] . These phagemid libraries are more easily handled and more stable than phage libraries, as the plasmid is incapable of causing phage production by itself. The libraries can therefore be more simply cloned, expanded, and controlled than phage libraries.

By infecting "helper" phage (based on M13) into a growing culture of E. coli harboring a phagemid library, the phage propagation machinery is provided, but due to a mutation introduced in the origin of replication on the helper phage genome, preferential packaging of the phagemid DNA and p3 fusion occurs during phage replication [ 8 ] . Phage production is thereby induced, genotype-to-phenotype linkage is created, and the expressed phage particles are interrogated for the presence of useful protein sequences via target binding. This selective step may be performed by simply immobilizing the target protein on, for example, a protein-binding plastic surface such as an ELISA plate, adding phage and allowing binding to occur via the antibody-p3 fusion proteins. Nonbinding phages are removed by washing the immobilized surface, and the remaining bound phages are eluted. The eluted phages are then reinfected back into a fresh culture of E. coli to retrieve the selected gene sequences. This process is not perfect, however, and two to four rounds of selection/re-expression/selection are normally performed iteratively to remove all unwanted clones and enrich the binding population from the background of the library. Nonetheless, this process can be spectacularly powerful, massively enriching specifi c antibodies in a single selection round [ 9 ] . Furthermore, the use of multiple forms of the target antigen in sequential selection rounds and the inclusion of competitor proteins can drive the selected pool toward a highly specifi c set of epitopes.

Much of the evolution and progression of phage display technology has been driven by the remarkable discovery that this method can be used to mine large libraries of combinatorial human antibody diversity, theoretically removing the need for animals in antibody production [ 10 ] . By random recombination of V H and V L sequences from human lymphocyte cDNA [ 10 ] , or, by using degenerate oligonucleotides to create diversity of DNA sequence in the loops associated with target binding [ 11 ] , several groups have created very large libraries of antibody gene sequences for phage display. These libraries are analogous to the naïve antibody repertoire in an animal, and selecting from them can result in the identifi cation of antibody fragments that exhibit high specifi city and occasionally high affi nity for the target protein. Several major studies have proven that with appropriate application of this technology, specifi c antibodies can be raised to proteins, peptides, haptens and even carbohydrates. In the most exemplary studies, antibodies have been raised with equivalent affi nities to those associated with a strong humoral immune response [ 12 -15 ] . Additionally, antibodies with suboptimal affi nities can provide useful starting molecules for the construction of mutagenized libraries which can be further screened for affi nity matured variants [ 16 -18 ] .

In addition to the affi nity maturation process, phage display also provides a useful tool for the production of ultra-humanized antibodies from nonhuman sources. It is widely accepted that there are intrinsic diffi culties associated with the humanization of nonhuman antibodies using CDR grafting. Frequently, such grafted antibodies suffer from immunogenicity associated with high non-germ-line amino acid content, the propensity for v-domain destabilization, and very often downstream expression and formulation issues. In a recent study, ultra-humanized antibodies from three nonhuman sources were generated via singlestep complementarity-determining region (CDR) germ-lining in a process termed " Augmented Binary Substitution" (ABS) [ 19 ] . This process utilizes phage display for selection from germline targeting combinatorial libraries which results in the identifi cation of CDR residues amenable to human germ-lining without compromising on specifi city and affi nity. A further degree of complexity is incorporated into the library through the random substitution of CDR-H3 residues in tandem with the germ-lining process. This simple single pass process generates signifi cantly lower non-germline sequence content, on CDR grafts from nonhuman hosts, rendering them virtually indistinguishable from fully human antibodies. This process opens up new possibilities for the affi nity maturation and humanization of antibodies from alternative sources such as chickens.

Recombinant antibodies are typically displayed and expressed in "fragment" forms. The simplest and most commonly used fragment is the single-chain fragment variable (scFv) where a fl exible peptide sequence links the V-regions of antibodies between the C-terminus of one domain and the N-terminus of the other, thereby combining both V-domains into a single polypeptide [ 20 ] . The scFv may be assembled in V L -V H or V H -V L orientations, with V H -V L being the most heavily used format historically. The fl exible linker helps to make the scFv simple to express, but must be sufficiently long and fl exible to allow effective association of the V-regions to form a functional antigen-combining site. As long as this is true, the classical hydrophobic pairing of the V-regions will stabilize the structure. By far, the most common linkers are based on glycine-serine repeat structures such as GGGGS × 3.

The second most commonly used recombinant antibody format is the Fab (fragment antigen binding) molecule. This structure is a complete binding "arm" of an antibody and is comprised of the full immunoglobulin light chain, expressed in conjunction with the V H -C H 1 region from the heavy chain [ 21 ] . Fabs obligately form predominantly monomeric, monovalent fragments. They are the most "natural" of the recombinant antibody fragments and it has been shown that the presence of the constant regions can often help to stabilize antibody variable regions [ 22 ] . The Fab format is the less commonly used of the two main recombinant antibody formats, however, as its dual polypeptide structure is generally more diffi cult to express and display in E. coli than the scFv [ 22 ] . A recent engineered scFab platform however attempts to address some of the limitations associated with scFab expression [ 23 ] .

Much of the under-use of phage display may be due to experiences with the early libraries derived from naïve or synthetic human antibody diversity, which were donated to academic laboratories that were not specifi cally invested in antibody engineering. Unfortunately, these forays into display technologies have often left investigators somewhat disappointed. Many people have accepted the viewpoints of recombinant antibody technology experts, that these libraries can yield useful high-affi nity antibodies to any form of antigen . In general, (mostly to those highly skilled in the fi eld), this is indeed true, but the average antibody generated from these libraries is often of disappointingly low affi nity to those who are used to high-sensitivity antibodies from immunized sources. Human recombinant antibodies from naïve library sources can require technically challenging in vitro molecular evolution if they are to perform the demanding "real world" functions required of many reagent antibodies [ 9 ] . Molecular evolution is far from trivial to perform and is usually beyond both the scope and interest level of the average researcher. For such reasons, it is of little wonder then that most people either ignore, or at worst disparage, phage display technology itself.

Nevertheless, phage display can be a relatively simple technology to use and when employed to harness natural repertoires of antibodies from immunized animals, it can offer a rapid path to highly specifi c, high-affi nity antibodies against problematic antigens . While the most successful naïve antibody libraries contain over 10 10 members and are often the domain of biotechnology companies, typical immune libraries are in the 10 7 -10 8 range and are easily assembled by a single investigator [ 24 , 25 ] . When an immunized rabbit or sheep has raised a signifi cant serum immunoglobulin titer, the common end-point to the experiment is to exsanguinate the animal and harvest the serum. However, harvesting B-cell rich lymphoid tissues from the animal, such as the spleen and bone marrow, allows the isolation of total RNA and the subsequent generation of cDNA [ 24 ] . This is a simple method with which many biomedical researchers are familiar, and commercial kits are available to simplify most steps of the process.

The immunoglobulin gene sequences of many animals are now known and the cDNA from immune tissues can subsequently be used for the RT-PCR amplifi cation and cloning of the animal's variable region sequence repertoire [ 24 ] . These cloned variable region sequences can then be assembled into a display library format such as scFv or chimeric Fab (using human C H 1 and C k/λ regions) [ 26 ] . These targeted immune libraries thereby offer a potentially huge advantage over monoclonal antibodies , as libraries of >10 8 variants may be built, allowing the effective sampling of a much broader range of antibodies than the hundreds (occasionally thousands) of clones usually examined in a monoclonal antibody screen.

The resulting library can be interrogated for specifi c binding proteins via phage display and the retrieved antibody fragments expressed very simply in bacteria [ 26 ] . This process has been used to successfully harness the antibody repertoires of a large number of immune host species, including mice [ 24 ] , rabbits [ 25 ] , sheep [ 27 ] , camelids [ 28 ], and sharks [ 29 ] . Of greater interest to us, however, is to exploit this approach to harvest the novel immunoglobulin repertoires of the domestic chicken ( Gallus gallus ), which is as simple to use as mice and rabbits, but also highly phylogenetically distant from mammals.

Avians can circumvent many of the common problems encountered with mammalian immunizations described above. As a fully domesticated small animal, chickens are an attractive host for immunization as they are highly accessible, very affordable, and easily housed in a generic animal house. Most importantly, however, the amino acid homology between the mammalian and avian orthologues of a given protein is typically lower than between the mammals commonly used for antibody generation, and indeed, some mammalian proteins may not even exist in avians. The immunoglobulin response of chickens to highly conserved mammalian proteins is reliably robust, generally exhibits high avidity, and potentially targets a broad spectrum of epitopes on protein immunogens [ 30 -32 ].

Chickens therefore have a potentially major advantage over other common immune hosts: they can produce a high-affi nity cross-reactive antibody response targeting an epitope that is conserved across multiple orthologues of a mammalian protein. This can lead to signifi cant savings in time and resources as, if a single, broadly applicable cloned reagent can be identifi ed, it can then be used to generate a single affi nity column for the capture of the target protein from multiple species. Chicken immunoglobulins have also shown benefi cial biophysical properties: they exhibit high stability to changes in pH and temperatures up to 70 °C [ 33 , 34 ] , provide functional coating on latex microspheres [ 35 ] , and demonstrate functional direct covalent coupling to a dextran layer for the detection of serum proteins by surface plasmon resonance [ 36 ] . Furthermore, as chickens are small animals, very little protein immunogen is required to raise a strong immunoglobulin response. Approximately 200 μg/bird of purifi ed protein is suffi cient to carry out a full immunization regime [ 37 , 38 ] .

These observations have led to the regular use of chickens as an immune host for production of the polyclonal antibody termed IgY (egg yolk antibody), in both research and commercial settings. Laying hens will export signifi cant quantities of polyclonal IgY into the egg (~100 mg of IgY per yolk), in a process analogous to mammalian placental IgG transfer, which allows direct screening of their antibody response without the need for serum sampling [ 38 ] . Once a strong immune response has been raised, large quantities of polyclonal antibody are easily prepared from the yolk. These polyclonal antibodies have been successfully applied in research immunochemistry [ 39 ], diagnostics [ 40 ] , and affi nity column purifi cation [ 41 ] . Indeed, immunodepletion resins based on chicken IgY can be used to remove high abundance proteins from serum and are now commercially available (Sigma-Aldrich Seppro ® IgY14 spin columns).

Unfortunately, polyclonal IgY does still suffer from the same issues of ill-defi ned specifi city that all polyclonal antibody preparations do. In addition, there have been several studies describing successful chicken hybridoma monoclonal antibody generation to antigens such as human peptides [ 42 ], sporozoite proteins [ 43 ], and prion protein [ 44 ], but the low antibody expression and instability associated with chicken myeloma cell lines [ 42 , 45 ] led to the under-use of this species as a source of monoclonal antibodies. Today however, the progress in chicken antibody phage display has circumvented these problems and made recombinant chicken antibody reagents readily accessible, as we describe below.

The chicken immunoglobulin repertoire is almost ideally suited to antibody phage display, as chickens generate their immunoglobulin repertoire from a single set of V H and V L germ line sequences [ 46 ] , with the majority of positions in the framework regions maintained as germline [ 2 ] . Diversity in the V-regions is created by both V-D-J recombination and somatic hypermutation, with the additional infl uence of "gene conversion," where multiple upstream pseudogenes are recombined into the functional sequence. This germline V-gene system means that the entire chicken antibody repertoire can be captured using only four PCR primers [ 47 ], making chicken libraries highly representative of the induced immunoglobulin response. This is in direct contrast with immune hosts such as mice, which have diverse germline V-gene sequences and therefore require complex mixes of PCR primers [ 24 ] . Additionally, the two V-gene germline sequences found in chickens are highly homologous to the human V λ and V H 3 germline families [ 48 ] , which are both associated with creating V-domains with high stability and solubility. Indeed, chicken scFvs can be stable in crude bacterial culture supernatants for up to 1 month at room temperature [ 49 ] .

The initial work of Davies et al. [ 47 ] showed that a simple recombinant chicken antibody library could be displayed on phage. While this small library was nonimmune and derived from the bursa cells of a single young chicken, the group was able to select target-specifi c scFv sequences recognizing lysozyme , serum albumin and thyroglobulin. The potential of chicken recombinant antibodies was further highlighted by a study [ 50 ] which used an scFv library derived from the spleens of immunized chickens and successfully generated highly specifi c scFv antibodies that targeted both mouse and rat serum albumins, where tolerance issues limit the ability to generate murine monoclonal antibodies . In addition, an in-depth analysis of the avian immunoglobulin V H repertoire demonstrated that naïve repertoire features were fully replicated in the resulting target-selected, phage display repertoire [ 2 ] .

A major study [ 26 ] subsequently demonstrated that chickens could be a useful source of scFv and chimeric Fab antibodies with specifi city for hapten molecules. However, none of these early studies characterized the antibodies for their affi nity, or their function as practical reagents. More recent studies have shown that scFv antibodies derived from immunized chickens are highly effective reagents in diverse settings such as diagnostic ELISA for Infectious Bursal Disease Virus [ 51 ], the diagnosis of prion disease [ 52 ], immunodetection of haptenic shellfi sh toxins [ 53 ], immunostaining of SARS-infected cells [ 54 ], biosensing of cardiac biomarkers [ 55 ] , and the measurement of ApoB protein in mouse and human sera [ 56 ] . Raats et al. [ 57 ] have also illustrated that antiidiotype scFvs from an immune chicken scFv library were of considerably higher sensitivity than those derived from a human

antibody library in the same study. The generation of highly selective scFvs toward the PrP protein, which is highly conserved in mammals demonstrates the advantage of the chicken as an immune model, as isolated scFvs were shown to react with murine, ovine, and bovine orthologues of the protein [ 52 ] .

The cloning of chicken antibodies via phage display has also allowed the precise dissection of the specifi city and affi nity of the chicken immunoglobulin response. High-throughput affi nity measurements for panels of chicken scFvs to the infl ammatory biomarker C-reactive protein have identifi ed clones that preferentially recognize the multimeric and monomeric forms of the protein [ 50 ]. In the same study, clones with affi nities as high as 350 pM were generated from an immune phage display library of only 3 × 10 7 total clones. In addition, chicken anti-PrP scFv have been reported to have affi nities up to 15 pM, making them among the highest affi nity scFvs reported to date [ 58 ] .

What may be of particular practical interest to many researchers is that chickens can serve as a host for simultaneous immunization with multiple proteins of interest, with as many as eight proteins being used successfully in a single immunization scheme [ 37 , 49 , 59 ] . The target proteins of interest are mixed in a single adjuvant preparation and each immunized animal receives all multiplexed proteins simultaneously. Spleen and bone marrow tissues from the immunized animals are then used to generate relatively small phage display libraries and specifi c antibodies are derived via selection of the library separately on the individual proteins originally used for immunization [ 37 ] . The immunized chickens appear to react to the proteins fully independently, as the phage display libraries generate individual scFv antibody clones that are fully specifi c by western blot and ELISA , showing no reactivity to their co-immunogens [ 37 , 59 ] . This approach has major benefi ts practically and ethically, as it allows the use of a single library to derive high-affi nity antibodies to a group of proteins of interest. Multi-immunization methods also simultaneously minimize animal use and raise the likelihood of success in generating an immediately useful reagent [ 37 , 59 ] .

Multi-target immunization regimes should be designed with one of two objectives in mind. Firstly, the simplest scenario combines multiple unrelated proteins, which leads to unrelated B-cell responses after immunization. To derive antibodies of greatest specifi city during a multi-target immunization of this kind, it is important to ensure that each of the protein immunogens is highly purifi ed and that no closely related proteins are co-immunized into a single animal. Secondly, to derive antibodies that are cross-reactive to orthologues of a conserved protein from multiple species, it is likely to be benefi cial, but not necessarily essential, to include each orthologue in the mix of immunogens given to each animal. Iterative selection rounds that change orthologue each time can then be used to bias toward the isolation of cross-reactive antibodies.

The generation and selection of chicken recombinant antibodies is extremely reliable using the methods described in detail in the accompanying chapter. The subsequent identifi cation and sequencing of antibodies displaying the characteristics desired can also be performed simply. In general, scFvs isolated from immunized chicken libraries exhibit high affi nity and can be assayed via a direct ELISA , using crude periplasmic extracts from the protein expressing E. coli clones. The level of further downstream analysis carried out on positive hits identifi ed during the binding ELISA depends on what the end user requires. Specifi c binding function may suffi ce for scFvs that are to be used simply as reagent antibodies for in vitro analysis of samples via ELISA or western blotting . For antibodies to be used in affi nity chromatography , the antibody fragments must be purifi ed and tested for their function after being coupled to a solid matrix and for their specifi city during purifi cation.

Few studies have been performed using antibody fragments in affi nity chromatography, but several potential approaches have been described. For the antibody binding site(s) to be fully solventexposed and active, the antibody fragment must be directionally captured onto the solid matrix. Even for full-length IgG , nonspecifi c adsorption or covalent coupling onto solid supports can lead to denaturation, reducing or negating antigen-binding function [ 60 ] . Antibody fragments may be slightly more prone to chemical or physical denaturation than full-length immunoglobulins , but scFv and Fab have been used successfully in affi nity chromatography, and in the creation of SPR sensing surfaces which can go through serial rounds of binding and regeneration [ 61 ] .

McElhinney et al. [ 62 ] created a simple scFv-based affi nity column for the concentration and cleanup of microcystin toxins from environmental samples, by transiently coupling the His-tagged scFv to a disposable nickel chelate column. The scFv was thereby coupled directionally, maximizing the functional antibody content on the column, and the analyte for purifi cation was co-eluted with the scFv before quantifi cation by reverse-phase HPLC. However, this method can only be used under a limited number of conditions, as the interaction of the His-tag with nickel is noncovalent and pH dependent. Other possibly useful low affi nity expression tags include E. coli maltose binding protein and glutathione S -transferase , which have both been successful in protein purifi cation [ 63 , 64 ] ( see also Chapter 8 for a discussion on protein tagging ).

High affi nity, highly stable linkage via affi nity tagging may also be achieved by site-specifi c biotinylation of antibody fragments and their immobilization onto a matrix that has been passively or covalently coated with avidin. Bacterial expression vectors are now available which introduce biotin into specifi c peptide tags ( AviTag ), which can be produced on the termini of recombinant proteins. A similar method was proven to be effi cient in the production of Fabs that are specifi cally biotinylated in vivo during bacterial expression, via C-terminal fusion of the Fabs to the E. coli acetyl-CoA carboxylase [ 65 ] . Importantly, these biotinylated Fabs were successfully used to purify recombinant TNF-alpha from bacterial lysates, via a streptavidinated column. The peptide tagging method has also been used successfully to label both Fab and scFv antibodies for their oriented immobilization and use as capture antibodies in clinical diagnostic ELISAs [ 66 , 67 ] . These studies suggest that biotinstreptavidin coupling is a simple and rapid method for the stable, directional capture of recombinant antibody fragments.

While the covalent coupling of recombinant antibody fragments via their reactive lysine side chains is likely to be disruptive to their function, some alternative covalent coupling methods have been identifi ed. In the simplest example, the disulfi de bonds linking the two constant regions of a Fab can be reduced using a mild agent to expose cysteine thiols. These thiol groups can then be used to covalently couple the fragment to a thiol-activated surface [ 68 ] . More elegant versions of this approach have expressed antibody fragments with a C-terminal cysteine group, then gently applied the same chemistry, to preferentially reduce the exposed disulphide groups [ 61 ] . The exposed terminal thiols are again an effi cient reactive group for covalent attachment. It is also possible to express scFv fused to the constant regions of human IgG light chains as another source of usable cysteine residues external to the V-regions [ 69 ] .

Whether any of the above attachment methods are appropriate for a given affi nity purifi cation application may be decided upon by the individual investigator. In cases where stable linkage has been achieved and the column is to be reused, it is prudent for the investigator to examine multiple clones for their stability under repeated cycles of elution and regeneration. While chicken scFvs are built upon naturally stable frameworks, the stability of different clones cannot be taken for granted. In cases where stability remains an issue, the appropriate chicken V-regions can be cloned into an Fc-fusion [ 70 ] or IgG [ 71 ] expression vector to produce full-length antibody in mammalian, yeast, and even plant culture systems [ 72 ] .

In conclusion, this chapter demonstrates that phage display is a powerful technology that can be used to generate highly specifi c reagents from immune sources. It is an attractive alternative to classical antibody generation technologies that can be employed in many fi elds for reliable antibody and antibody fragment generation .

Antibody microarrays: promises and problems

Fundamental characteristics of the immunoglobulin VH repertoire of chickens in comparison with those of humans, mice, and camelids

Analyzing antibody specifi city with whole proteome microarrays

Continuous cultures of fused cells secreting antibody of predefi ned specifi city

Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface

Cloning immunoglobulin variable domains for expression by the polymerase chain reaction

Phage antibodies: fi lamentous phage displaying antibody variable domains

Multi-subunit proteins on the surface of fi lamentous phage: methodologies for displaying antibody (Fab) heavy and light chains

Selecting and screening recombinant antibody libraries

Human antibodies from V-gene libraries displayed on phage

Semisynthetic combinatorial antibody libraries: a chemical solution to the diversity problem

Human antibodies with sub-nanomolar affi nities isolated from a large non-immunized phage display library

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

Generation of high-affi nity human antibodies by combining donor-derived and synthetic complementarity-determiningregion diversity

High-throughput generation of synthetic antibodies from highly functional minimalist phage-displayed libraries

Picomolar affi nity antibodies from a fully synthetic naive library selected and evolved by ribosome display

Affi nity maturation of a humanized rat antibody for anti-RAGE therapy: comprehensive mutagenesis reveals a high level of mutational plasticity both inside and outside the complementarity-determining regions

In vitro selection and affi nity maturation of antibodies from a naive combinatorial immunoglobulin library

Augmented binary substitution: single-pass CDR germ-lining and stabilization of therapeutic antibodies

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

Human monoclonal Fab fragments derived from a combinatorial library bind to respiratory syncytial virus F glycoprotein and neutralize infectivity

Domain interactions in the Fab fragment: a comparative evaluation of the singlechain Fv and Fab format engineered with variable domains of different stability

An improved single-chain Fab platform for effi cient display and recombinant expression

Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system

High affi nity ScFvs from a single rabbit immunized with multiple haptens

Methods for the generation of chicken monoclonal antibody fragments by phage display

The isolation of super-sensitive anti-hapten antibodies from combinatorial antibody libraries derived from sheep

High throughput ranking of recombinant avian scFv antibody fragments from crude lysates using the Biacore A100

Determination of LOX-1-ligand activity in mouse plasma with a chicken monoclonal antibody for ApoB

Generating recombinant anti-idiotypic antibodies for the detection of haptens in solution

Humanization of chicken monoclonal antibody using phagedisplay system

Multipleantigen immunization of chickens facilitates the generation of recombinant antibodies to autoantigens

Profi ling receptor tyrosine kinase activation by using Ab microarrays

Oriented immobilisation of engineered single-chain antibodies to develop biosensors for virus detection

Rapid isolation of a single-chain antibody against the cyanobacterial toxin microcystin-LR by phage display and its use in the immunoaffi nity concentration of microcystins from water

Advances in recombinant antibody microarrays

Comparison of affi nity tags for protein purifi cation

In vivo biotinylated recombinant antibodies: construction, characterization, and application of a bifunctional Fab-BCCP fusion protein produced in Escherichia coli

In vitro enzymatic biotinylation of recombinant fab fragments through a peptide acceptor tail

Use of an in vivo biotinylated single-chain antibody as capture reagent in an immunometric assay to decrease the incidence of interference from heterophilic antibodies

Sitedirected immobilisation of antibody fragments for detection of C-reactive protein

Recombinant antibody expression vectors enabling double and triple immunostaining of tissue culture cells using monoclonal antibodies

Production of anti-prion scFv-Fc fusion proteins by recombinant animal cells

Whole IgG surface display on mammalian cells: application to isolation of neutralizing chicken monoclonal anti-IL-12 antibodies

Plant expression of chicken secretory antibodies derived from combinatorial libraries