@franguzzo said:

l'hai tolto appena in tempo il ditino medio, stava per arrivare GameOver

Era sprecato con te non trovi???

@franguzzo said:

Neanche nella trilogia del Signore degli Anelli ho mai sentito una frase del genere

Per inciso : non mi interessa e non mi rende felice vincere un campionato a tavolino, il post l'ho messo unicamente per fare un po' di spettacolo ed avanspettacolo ed al massimo per qualche sana e reciproca presa per i fondelli!!

Poi, però, vedendo certe frasi deliranti, mi viene quasi voglia di festeggiare davvero!!

BRAVO. TIENI DURO.

@sitionweb said:

Assolutamente non sono d'accordo.

Grazie Triade di che? DI averci fatto finire in serie B? Di averci reso la **vergogna **del calcio italiano e mondiale? Di aver macchiato l'onore e lo stile Juve (quello degli Agnelli dei bei tempi...)?

Se è tutto questo, allora grazie triade, ma ve lo potevate risparmiare.

Non capisci...la TRIADE ci ha difeso..dal potere occulto che esercitavano altre forze di cui non sto a parlare qui...l'unico modo per difenderci era attaccare..e loro lo hanno fatto...e ci hanno resi la squadra più forte per anni..e anni...

NOn dimenticare le gioie del passato e i momenti belli che ti hanno regalato..

GRAZIE LUCIANO MOGGI. GRAZIE GIRAUDO. GRAZIE BETTEGA( anche se non so c. hai fatto....) NON VI DIMENTICHEREMO MAI...

GRAZIE PER AVERCI FATTO AIUTATO A DIMOSTRARE A TUTTI CHE LA JUVE E' LA PIU' FORTE ED E' UNA SOCIETA' VINCENTE.

PURTROPPO QUANDO LE REGOLE SONO "SPORCHE" O TI ADEGUI..O NON GIOCHI...O SEI L'INTER...

NOI SIAMO LA JUVE...VINCENTI...DENTRO E FUORI....

A TUTTI QUELLI CHE LEGGENDO LE MIE PAROLE NELL'ANIMO SANNO CHE HO RAGIONE DICO: ASPETTATE...RITORNEREMO....E PER VINCERE...ANCHE SENZA TRIADE...

Ora basta, abbandono questo topic per non rovinare la festa a franguzzo & c.

HAI VINTO LO SCUDETTO!!!!!!!!! GRANDE!!!!!!!!!!! VAI A FARE CAROSELLO!!!!!!!!!!!!!!!!! VAI............ VAI............... VAI............COMPLIMENTI

@sitionweb said:

Potrei essere d'accordo con te nel non assegnarlo

Ma l'Inter è una squadra assolutamente pulita ed onesta. E sinceramente preferirei essere onesto e non vincere mai nulla che vincere imbrogliando.:

Io no, il fine giustifica i mezzi: GRAZIE TRIADE.

[/QUOTE]E poi, una piccola considerazione: siamo proprio sicuri che, se non ci fossero stati gli imbrogli, l'Inter non avrebbe mai vinto nulla?[/QUOTE]
Assolutamente no. Quando sei un perdente, sei un perdente. Se ci mandavano tutti in B ti assicuro che l'Inter non avrebbe vinto lo scudetto lo stesso...sono dei perdenti punto e basta.

[/QUOTE]Te lo ripeto: sono juventino. E da juventino, mi viene da chiedermi: come mai la juve ha quasi 30 scudetti e solo 2 Champions? Forse che all'estero il cellulare non abbia tanta copertura? [/QUOTE]

Purtroppo si...

@franguzzo said:

a leggere la tua reazione scomposta e aggressiva, mi vergogno io per te
Non vedo nulla di aggressivo ne di scomposto...solo una constatazione...
Torna a fare i giochini franguzzo che è meglio...

Ma esultare di cosa??? Cosa c'è da esultare??? Lo scudetto l'ha vinto la juve e se glielo vogliamo togliere la decisione giusta è NON ASSEGNARLO...ma hanno deciso di fare di più...per ironizzare contro gli interisti..che fanno ridere l'Italia per l'incapacità di una squadra di spendere miliardi su miliardi per i migliori giocatori e poi non vincere un c.

Per me siete dei perdenti, dentro e fuori. Esultando pure mi sembrate altresì patetici e privi di vergogni.

Non ho parole...siete veramente dei perdenti dentro!!! Avete addirittura il coraggio di esultare ad una sentenza che sottolinea ancora di più ( e a mio parere ironizza pure..) sul fatto che siete una squadra di m.
Ormai gli interisti della sconfitta ne hanno fatto la loro bandiera..e ormai ci si trovano a loro agio...

L'unico modo che avevate di vincere qualcosa era questo...peccato che non ci sia lo stesso gusto che a vincerle in campo....

Vedervi esultare mi fa vergognare per Voi...

Eccomi qui...

TEST BENEXOL.

Alcolici bevuti( naturalmente per testare il farmaco)
1 jaegermeister
3 Mojito
4 rum & fruit

Rimedi
2 litri d'acqua + 2 pastiglie benexol

Ore dormite
3

Day-after
mal di testa: 0
stanchezza fisica: media
mal di stomaco:0

Fantastico...potrei bere tutte le sere....:)

@ludus said:

io risolvo in maniera molto intelligente: non mi sono mai ubriacato e non ho intenzione di farlo in futuro

Esagerato...una sbronza una volta ogni tanto merita di essere presa...ripeto...una volta ogni tanto..

Io reggo bene..spesso vado a letto alle 4 con la testa che gira e alle 7,30 sono già in piedi diretto per l'ufficio. Fuori non si vede niente ( e tutti mi dicono che è una fortuna immane...) ma dentro....

Ho trovato un topic sul web
[url=http://www.covodeglisbronzi.it/apubslife.php?id_art=54]A Pub's Life
[url=http://www.maleizappa.it/blog/blog_commento.asp?blog_id=20&month=8&year=2005&giorno=&archivio=]MALEIBLOGGA - Il Blog dei Maleizappa!

Cmq l'ho comprato 15 minuti fa...stasera lo provo..domattina vi dico!!!!!

P.s. sul primo topic l'attendibilità e da verificare ma sulle competenze mediche del dott. VICTOR PUTTANONE non penso ci siano dubbi....

@Dusy said:

almeno con me ha sempre funzionato...

Forse ha funzionato nel farti diventare un alcolizzata

Qualcuno conosce gli effetti del Benexol per i postumi della sbornia?
Della Bayer.

Se qualcuno lo usa/lo ha usato sarei curioso di sapere il parere...

Grazie

Phage display: practicalities and prospects

William G.T. Willats

Department of Biochemistry and Molecular Biology; University of Leeds, Woodhouse Lane, LS2 9JT, UK (Tel: +44 (0)113 3433168; Fax: +44 (0)113 3433144; E-mail: [email][email protected][/email])

Accepted 20 August 2002

Key words: antibody microarrays, immunomodulation, molecular evolution, Phage display, protein interactions, recombinant antibodies

Abstract

Phage display is a molecular technique by which foreign proteins are expressed at the surface of phage particles. Such phage thereby become vehicles for expression that not only carry within them the nucleotide sequence encoding expressed proteins, but also have the capacity to replicate. Using phage display vast numbers of variant nucleotide sequences may be converted into populations of variant peptides and proteins which may be screened for desired properties. It is now some seventeen years since the first demonstration of the feasibility of this technology and the intervening years have seen an explosion in its applications. This review discusses the major uses of phage display including its use for elucidating protein interactions, molecular evolution and for the production of recombinant antibodies.
Abbreviations: scFv ? single chain variable fragment of an antibody; dsFv ? disulphide stabilised scFv; Fab ? antigen binding antibody fragment; PCR ? polymerase chain reaction; RG II ? rhamnogalacturonan II; CDR ? complimentarity determining region; VH ? variable region of an antibody heavy chain; VL ? variable region of an antibody light chain; HG ? homogalacturonan; AceHG ? acetylated HG; ELISA ? enzyme linked immunosorbent assay; GFP ? green fluorescent protein.
Introduction or antibody fragments at the surface of phage par¬
ticles (Smith, 1985; Winter et al., 1994; Kay and Phage display technology has had a major impact on Hoess,1996). This is accomplished by the incorpora¬immunology, cell biology, drug discovery and phar-tion of the nucleotide sequence encoding the protein macology and is increasingly gaining importance in to be displayed into a phage or phagemid genome as plant science. The aim of this review is to provide a fusion to a gene encoding a phage coat protein. This a practical survey of the principles and applications fusion ensures that as phage particles are assembled, of phage display. The emphasis will be on the rela-the protein to be displayed is presented at the surface tive merits of this technology for addressing diverse of the mature phage, while the sequence encoding it is biological problems and the practicalities of what is in-contained within the same phage particle (Figure 1a volved, rather than a detailed exposition of molecular and 1b). This physical link between the phenotype technique or a comprehensive review of the literature. and genotype of the expressed protein and the replica-
Phage display is an extremely powerful tool for tive capacity of phage are the structural elements that selecting peptides or proteins with specific binding underpin all phage display technology (Figure 1b). properties from vast numbers of variants. Its utility Using phage display, libraries of variant nucleotide lies principally in generating molecular probes against sequences with diversities of millions or billions may specific targets and for the analysis and manipulation be converted into populations of displayed variant pro¬of protein/ligand interactions. Put at its most simple, teins which can then be conveniently screened for phage display is the expression of peptides, proteins

Figure 1. The principle of phage display. (a) The simplified hypothetical bacteriophage shown has a genome that contains an origin of replication (Or) and genes (g1 and g2) that encode two types of coat protein -p1 and p2, respectively. A foreign protein pχ, that is encoded by gene gχ, may be displayed at the phage surface by the fusion of gχ to one of the phage coat protein genes. The number of copies of pχ displayed is related to which phage coat protein (p1 or p2) is chosen as a fusion partner. (b) This principle can be applied to the expression of natural or random peptides, protein domains or whole proteins and antibody fragments. (c) Using phage display, a library of variant nucleotide sequences can be converted into a library of variant peptides or proteins. The phage display library may then be screened in order to isolate phage displaying peptides or proteins with desired properties.

desirable properties (Figure 1c). Screening of phage display libraries is usually accomplished by an affin¬ity selection (or bio-panning) process during which phage populations are exposed to targets in order to selectively capture binding phage (Hoogenboom, 1997; Hoogenboom et al., 1998; Sparks et al., 1996). Throughout successive rounds of binding, washing, elution and amplification, the originally very diverse phage population is increasingly enriched with phage with a propensity to bind to the target in question. Ul¬timately, monoclonal phage populations with desired specificities can be selected. This procedure of DNA manipulation to create a library of variants, packaging into phage, and subsequent bio-panning is the basic protocol for all phage display and has been coined the ?phage display cycle? (Hoogenboom et al., 1998) (Figure 2). Because the genotype of each protein phe¬notype is carried within phage particles, once proteins of interest have been isolated the sequence encoding them can be readily determined and altered in order to manipulate or refine binding properties.
Phage display WWW resources
A wealth of information and physical resources relat¬ing to phage display is now available via the World Wide Web. Table 1 is a list of the URLs for some of the major sources of phage display libraries and information.
Why use phage display?
As a system for the high throughput analysis of protein interactions phage display is complimentary to, rather than a substitute for, other methods such as yeast hy¬brid systems (Drees, 1999; Mendelsohn and Brent, 1999; Uetz, 2001) (see also pages X ? X of this issue) and each have their advantages and limitations. One advantage of phage display is the enormous diversity of variant proteins that can be represented. For exam¬ple, phage display antibody libraries with diversities as high as 1010 are routinely constructed (Hoogen¬boom et al., 1998) (see also Section 7). Phage display is highly flexible and selection may be performed in vivo or in vitro. (Johns et al., 2000; Sparks et al., 1996; McCafferty and Johnson, 1996). In vitro selec¬tion enables phage displayed proteins to be screened not only against a wide range of biological targets but also inorganic ones (Whaley et al., 2000). Yeast hybrid systems have the potential advantage that protein in¬teractions are assessed under physiological conditions ? but only a limited range of conditions are avail¬able, namely those within yeast cells (Drees, 1999). In contrast, phage display screening formats can be read¬ily modified to manipulate selection conditions and stringencies (Watters et al., 1997; Rodi et al., 2001; Hoogenboom et al., 1998). Both yeast hybrid systems and phage display provide a means of rapidly screen¬ing large numbers of proteins against potential binding partners but phage display has the higher throughput. As a rough guide, billions of clones can be screened within a week using phage display while with yeast two-hybrids millions of clones can be screened in two to four weeks (Rodi et al., 2001). An attractive aspect of phage display is that provided appropriate libraries can be obtained (Table 1), the technique is simple, cheap, rapid to set up and requires no special equipment.

Phage Display vehicles
The two key physical elements of phage display are the libraries of nucleotide sequences encoding peptides or proteins (e.g. gene fragments, random oligonucleotides, cDNA populations or antibody gene sequences) and the phage vehicles on which these se¬quences are expressed. The simplest way of achieving the expression of a foreign protein is to simply cre¬ate a fusion between the nucleotide sequence to be expressed and a coat protein gene within the viral genome (Figure 3a). Using this direct approach all the copies of the chosen coat protein become fusion pro¬teins (Winter et al., 1994). This can be advantageous in terms of numbers of expressed foreign proteins but if the functionality of the chosen coat protein is com¬promised by the fusion then phage viability may be affected, especially since no wild type versions of the coat protein are retained. This is avoided if hybrid phage are produced in which some versions of a given coat protein are wild type and some are fused to a for¬eign protein (Figure 3b and 3c). In some hybrid phage systems the gene fusion is an additional element of the phage genome so that a wild type copy of the coat protein gene is retained and phage particles express both wild type and fusion proteins (Figure 3b) (Sidhu, 2001). Alternatively, hybrid phage may be created us¬ing a phagemid-based system and this approach has been widely adopted (Figure 3c). Sequences encod¬ing fusion proteins are carried by phagemids (plasmids with a phage origin of replication) while the majority of the genes required for the formation of phage parti¬

Figure 2. The phage display cycle (a) A library of variant DNA sequences encoding peptides or proteins is created and (b) cloned into phage or phagemid genomes as fusions to a coat protein gene (see also Figure 3). (c) The phage library displaying variant peptides or proteins is exposed to target molecules and phage with appropriate specificity are captured. (d) Non binding phage are washed off ? although some non-specific binding may also occur. (e) Bound phage are eluted by conditions that disrupt the interaction between the displayed peptide or protein and the target. (f) Eluted phage are infected into host bacterial cells and thereby amplified. (g) This amplified phage population is in effect a secondary library that is greatly enriched in phage displaying peptides or proteins that bind to the target. If the bio-panning steps (c) to (f) are repeated the phage populated becomes less and less diverse as the population becomes more and more enriched in the limited number of variants with binding capacity. (h) After several (usually three to five) rounds of bio-panning monoclonal phage populations may be selected and analysed individually.

Table 1. Phage display resources on the WWW.

Source URL Comments
Smith Lab. University of Missouri www.biosci.missouri.edu/smithgp/Pha- A source of peptide phage display libraries
geDisplayWebsite/ PhageDisplayWebsite¬ and associated information
Index.html
MRC Centre for Protein Engineer¬ www.mrc-cpe.cam.ac.uk/∼phage/ The Winter group has previously provided
ing. The Winter Group home page. synthetic antibody libraries. Although not
distributed at present, copies of these li¬
braries may be obtained from existing users.
This site also contains useful phage display
information.
S. Dübel, at the University of Hei¬ www.mgen.uni- A comprehensive source of recombinant an¬
delberg, Molecular Genetics heidelberg.de/SD/SDscFvSite.html tibody resources
The Queen?s University of Belfast www.qub.ac.uk/bb/awpage/faq.htm Extensive information about obtaining phage
display libraries and associated reagents.
General information about phage display
protocols
New England BioLabs www.neb.com/neb/frame_cat.html Information about the PhDTM phage display
peptide libraries as well as useful general
phage display information
MRC Centre for Protein Engineer¬ www.mrc-cpe.cam.ac.uk/imt- A database of human antibody genes
ing. V Base (MRC) doc/public/INTRO.html
Philipps-Universität Marburg http://aximt1.imt.uni- Phage display and general filamentous phage
marburg.de/ rek/AEPphage.html information
University of Nijmegen http://baserv.uci.kun.nl/ jraats/links1.html Comprehensive phage display links

cles are carried by helper phage that are co-infected together with phagemids into host bacteria (Sidhu, 2001) (Figure 3c).
Hybrid phage systems have the potential disad¬vantage that the average number of displayed fusion proteins is reduced because of competition for incor¬poration into the phage particle between wild type and fusion coat proteins (Winter et al., 1994; McCafferty, 1996). However, low valency can be used as a strategy to select for high avidity binders during bio-panning. If coat protein functionality is not completely com¬promised by fusion to a foreign protein, then valency can be increased in phagemid systems by the use of modified helper phage (such as M13gIII) that lack the gene for the chosen coat protein (Winter et al., 1994; Rondot, 2001; Griffiths et al., 1993). More¬over, the choice of coat protein fusion partners has been extended recently by the development of new mutant variants of coat proteins and even completely artificial coat proteins (Sidhu, 2001). The number of expressed proteins therefore depends on the coat pro¬tein chosen as a fusion partner, the display system used (phage or phagemid) and, if a phagemid system is used, the choice of helper phage. A refinement of some phage display systems is the insertion of an am¬ber stop codon between the sequences encoding the coat protein and the displayed foreign protein. This allows a soluble (i.e. non-phage bound) version of the foreign protein to be produced if the phage are propa¬gated in an appropriate non-suppressing strain of host bacteria (Winter et al., 1994). Peptide tags such as c¬myc and poly-histidine are routinely incorporated into displayed proteins for ease of subsequent purification and detection.
Many types of phage have been used as vehi¬cles for phage display including Ff filamentous phage, Lambda and T7 (Rodi and Makowski, 1999; Danner and Belasco, 2001). Each of these has advantages and disadvantages with respect to each particular applica¬tion. The Ff phage family (M13 and its close relatives fd and fl) are excellent cloning vehicles because their size is not constrained by the DNA contained within them. The insertion of foreign sequences within their genome is accommodated simply by the assembly of longer phage particles. On the other hand, the non-lytic propagation mechanism of Ff phage requires that

Figure 3. Phage display formats (a)?(c) Strategies for the expression of proteins at the surface of a simplified hypothetical bacteriophage (see also Figure 1). (a) The simplest format for the expression of a peptide or protein is to fuse the gene (gχ) encoding the foreign protein (pχ) to one of the phage coat protein genes (e.g., g1) (see also Figure 1). This strategy produces phage particles in which all the copies of chosen phage coat protein are fusion proteins (p1/pχ). (b) Hybrid phage may be created by incorporating the gene fusion (g1/gχ) as an additional element in the phage genome. With this arrangement, two versions of the phage coat protein chosen as the fusion partner are encoded -one by the native gene (p1) and one by the fusion gene (p1/pχ). As phage particles are assembled both p1 and p1/pχ are incorporated into the phage coat. (c) Phagemid based systems are also widely used to construct hybrid phage. However, instead of being present on a single genome, the genes encoding wild type coat protein and fused protein are carried by helper phage and phagemid respectively. Host bacteria contain both phagemid and helper phage DNA and both genomes contribute to the synthesis of hybrid phage particles. (d) M13 bacteriophage are widely used as vehicles for phage display. The pIII coat protein can be used as a fusion partner for a limited number (maximum of five) of proteins while thousands of proteins can be expressed at the phage surface if pVIII is used as a fusion partner. The approximate number of copies of each M13 coat protein is indicated.
the all the components of the phage coat be exported proteins at high density (Zucconi et al., 2001). In some through the bacterial inner membrane prior to the cases it may even be advantageous to combine dif¬assembly of the mature phage particle. As a conse-ferent phage types in one experiment. This approach quence, only proteins that are capable of withstanding was used by Castillo et al. (2001) in order to select this export may be displayed (Danner and Belasco, anti-peptide single chain antibody fragments (scFvs). 2001). This limitation may be avoided by using the Whilst the peptide targets were displayed on T7, the lytic phage Lambda and T7, in which capsid assembly scFvs were selected from an M13 display library. occurs entirely in the cytoplasm prior to cell lysis. Fur-Despite some limitations, the Ff bacteriophage thermore, recent studies have shown that unlike T7, provide a robust and highly flexible platform for dis-Lambda phage can tolerate the display of relative large play and have been widely adopted. These long (about 1 µm) phage particles consist of single stranded DNA packaged into a coat consisting of five different types of coat protein, all of which have been used for the display of foreign proteins (Figure 3d) (Sidhu, 2001; Hoogenboom et al., 1998; Rodi and Makowski, 1999). Each of the coat proteins have their relative merits as fusion partners with respect to the number of fusion proteins displayed per phage, the effects of expressed fusion proteins on phage viability, and stability of the fusion proteins (Sidhu, 2001; Rodi and Makowski, 1999). In general terms, large numbers of smaller pro¬teins may be displayed if pVIII is chosen as a fusion partner, whilst pIII is a suitable partner for smaller numbers of larger proteins.

Finding the needle in the haystack: screening phage display libraries
Once a phage display library has been constructed or acquired the task is to screen the library in such a way that the original very high diversity of the library is reduced to a manageable number of clones which can then be analysed in detail. Most screening procedures are based on affinity selection and involve the follow¬ing fundamental steps: 1, A library is amplified and phage particles produced; 2, phage particles are ex¬posed to a target for which a binding protein is sought; 3, non-binding phage are removed by washing and 4, binding phage are eluted, infected into host bac¬teria and thereby amplified. These bio-panning rounds are then repeated, typically three to six times. In the following sub-sections some general considerations involved in the various steps of phage display library screening are discussed.
Library amplification
Although libraries with very high diversities are avail¬able, some expressed sequences are incompatible with phage propagation, whilst others are highly suscepti¬ble to proteolysis during propagation. These factors impose constraints on effective diversity and it is therefore desirable to start with a library that is as diverse as possible (Sparks et al., 1996). The possibil¬ity that some expressed sequences may be somewhat deleterious to phage propagation can be militated to some extent by including a growth step in each pan¬ning round that creates less competitive growth condi¬tions, for example by growing on solid media rather than exclusively in liquid culture. It is also important to empirically check the diversity of libraries before starting any screen because of the possibility that in¬stabilities in libraries can lead to loss of inserts. A quick check of diversity can be made by simply plating out a representative portion of the library, selecting a number of individual clones and then using PCR to check what proportion of clones contain inserts.
Bringing phage and targets together
One of the strengths of phage display is that screen¬ing protocols can readily be tailored to the particular requirements of many different target molecules. The simplest and most widely used approach is to immo¬bilise target molecules to a support and then to expose solutions containing phage to the immobilised target. Many variations to this theme have been successfully used including immobilisation onto coated tubes or plates, within columns or on the surface of magnetic beads. Immobilisation of many bio-molecules can be achieved by passive adsorption onto polystyrene tubes with an appropriate surface modification such as MaxiSorpTM (Nissim et al., 1994). Passive adsorption has the convenience that a wide range of molecules can be immobilised without any prior treatment. How¬ever, passive adsorption relies on establishing a large number of relatively weak bonds between target and support which can result in the immobilised mole¬cule being forced out of its functional configuration (Wilson and Nock, 2001). Clearly, this is undesirable if protein ligands are sought to functional versions of targets. A solution to this is to create one, or a limited number of tighter interactions between sup¬port and target ? for example by using biotinylating targets (Hoogenboom et al., 1998). More innovative screening methods have also been employed includ¬ing panning against whole fixed or living cells, tissue sections or even within living animals (Watters et al., 1997; Johns et al., 2000). Screens may also be de¬signed such that specific complexes can be selected for. One example of this is infectivity screening based on phage bearing truncated, non-infective fusion coat proteins. Infectivity is restored only if a complex is formed with a binding partner that has the capacity to restore infective functionality to the truncated coat protein.
Washing and elution
The basic purpose of washing is to remove non¬binding phage from the selection process so that binding phage are selectively enriched. However, this step is worth some consideration because a balance is required between specificity and avidity of selected clones. Most phage display libraries of whatever sort are likely to contain clones with a spectrum of avidities for any particular target. Some may be strong binders with low specificities, others the reverse. If washing is too stringent then highly specific, but weak binders may be lost. If washing is not stringent enough then populations of selected clones may be dominated by strong binders with low specificity. In practice this bal¬ance is achieved by adjusting washing times, detergent concentrations and using regimes in which washing stringencies are progressively increased. A number of treatments can be used to elute bound phage from tar¬gets. Dramatically lowering or increasing pH is often employed, or reducing agents may be used to disrupt disulphide-based links between supports and targets. A more subtle approach using enzymatic cleavage can be used where there are concerns about the effects on phage integrity of harsh elution conditions. Enzyme cleavage sites can be incorporated into the fusion pro¬tein, for example a trypsin cleavage site can be inserted between M13 pIII and the displayed fusion protein (Rondot et al., 2000).
Re-infection into host cells
It is usually assumed that following elution of bound phage, it is always essential to then amplify the re¬covered phage population before the next round of bio-panning, and indeed virtually all protocols include this step. However, this dogma may be worth careful examination since some reports indicate that directly using eluted phage without amplification may reduce background problems and help reduce the number of non-specific phage that are inevitably carried through the panning process. The rational is that during am¬plification, phage with inferior avidities for the target but better growth characteristics may be preferentially amplified. This has some important practical implica¬tions. The in vivo amplification steps are the most time consuming part of phage display library screening and if they could be avoided the time required for each screen would therefore be greatly reduced. Moreover, without the in vivo steps it is much easier to envisage how the whole screening process could eventually be completely automated (Hoogenboom et al., 1998).
What can be expressed at the surface of phage?
The first incarnation of phage display involved the en¬richment of just one expressed protein against a wild type phage population (Smith, 1985). In the inter¬vening seventeen years the scale and scope of phage display has vastly increased. Natural and synthetic peptides, proteins and protein domains and synthetic antibodies are now all routinely displayed on phage (Winter, 1998a; Winter et al., 1994; Kay and Hoess, 1996).

Phage display of peptides and proteins
The starting point of much peptide phage display work is the generation of random combinatorial libraries that provide a pool of variants from which peptides can be isolated by affinity selection. The peptides dis¬played in these libraries typically range in length from 5 to 20 amino acids and in some cases the conforma¬tional flexibility of displayed peptides is constrained by cyclisation. This is likely to afford some protection against proteolysis and may yield peptides with higher affinities. Cyclisation can be achieved by an amide bond between the N-alpha group and the side chain of the last residue or by a disulphide bridge between cysteine residues positioned at the N-and C-termini. A number of peptide libraries are freely available from the Laboratory of George Smith, University of Mis¬souri (Table 1). Random peptide libraries are a source of binding partners for a wide range of targets and in some cases the objective of phage display is to sim¬ply use isolated peptides directly as molecular probes or agonists. However, peptides may also be isolated with sequence homology to the natural protein bind¬ing partners of targets and such ?convergent evolution? studies are a powerful application of peptide phage display (Kay and Hoess, 1996).
Convergent evolution
The theory of convergent evolution of peptides is that by affinity selection, peptides can be isolated from a diverse starting pool that interact with a given target. Furthermore, the isolated peptides may have sequence homology to the natural binding partners of the target. Therefore, if the genome of the organism in ques¬tion has been sequenced to a significant extent, then the sequences from selected phage displayed peptides can be used to identify their natural counterparts by

Figure 4. Convergent evolution The natural protein binding partners of a given target may be identified by isolating phage displayed peptides that bind to the target and comparing them to a database of native sequences. (a) A population of variant nucleotide sequences are package into phage to generate a phage library displaying variant peptides (b). (c) Phage are screened against a target and binding peptides isolated (d). (e) A database for the organism in question is then searched for sequences with homology to the sequences encoding peptides carried by binding phage. Genes containing sequences with such homology may then be considered as candidates that encode the natural binding partner of the

target.
homology comparison (Kay et al., 2000) (Figure 4). One obvious danger is that such screens will isolate not peptides with homologous sequences, but ?mimo¬topes? -peptides that bind just as tightly to the target as the natural binding partner, but have no resem¬blance to it at the sequence level. However, various screening strategies have been developed to minimise this outcome (Rodi and Makowski, 1999) and conver¬gent evolution has proved to be a powerful strategy for unravelling protein interaction networks. Never¬theless, it perhaps seems surprising that short peptides can mimic so closely the interactive characteristics of often much larger and more complex natural counter¬parts. The explanation lies in the fact that for many proteins only a small subset of residues account for most of the change in free energy that mediates bind¬ing. For example human growth hormone consists of 217 resides but eight of these account for 85% of the binding energy (Rodi and Makowski, 1999).
Directed evolution
Once a population of peptide or protein ligands has been isolated, further layers of modification and selec¬tion can be applied in order to enhance or manipulate binding properties or affinities (Figure 5). The strategy of directing a population of peptides or proteins to¬wards specific properties by creating random sequence variation is known as directed evolution. In contrast to rational approaches for manipulating the properties of proteins, directed evolution has the advantage that pro¬teins can be manipulated without the need for a prior knowledge of molecular structure, or of the details of molecular action. Using directed evolution it has been possible to identify stronger binding ligands to recep¬tors, and to produce novel enzyme inhibitors and DNA binding proteins (Lowman and Wells, 1993; Dennis and Lazarus, 1994; Choo et al., 1994). The products of convergent evolution experiments can be a fruitful source of variants upon which further diversity can be imposed. Using the sequences encoding isolated peptides as a starting point, a second combinatorial library may be generated that is varied around selected sequences. The starting point for directed evolution can also be a protein of which the function is already known and characterised. A number of strategies are employed to introduce limited variation, including er¬ror prone PCR, the amplification of phage populations in mutator strains of host bacteria and DNA and fam¬ily shuffling. A recent example of this approach is the creation of variant forms of phytocystatin protease inhibitors (McPherson and Harrison, 2001). It has been demonstrated that protease inhibitors expressed in plants under the control of appropriate promoters can confer resistance to plant parasitic nematodes. Of the more than 60 phytocystatin sequences now known, eleven were chosen and subjected to family shuffling. The library of variants is now being screened with the intention of isolating phytocystatins with more potent inhibitory characteristics.

Figure 5. Directed evolution Phage display is a powerful tool for molecular evolution. A phage library displaying peptides or proteins is screened against a target and the binding properties of selected peptides or proteins are assessed by an appropriate assay. The nucleotide sequences encoding the selected peptides or proteins are then altered, for example by error prone PCR or DNA shuffling to create a new population of variant nucleotides. The phage display, bio-panning and analysis steps are then repeated in the hope of finding peptides or

proteins with altered or improved binding properties.
Directed evolution is also an important tool for the manipulation of enzyme characteristics and displayed variants of a given enzyme may be rapidly screened for altered properties. This approach is illustrated by the selection of lipase variants from an M13 phage library (Danielsen et al., 2001). Nine amino acids close to the active site of lipase from Thermomyces lanuginosa were targeted for randomisation by cassette mutage¬nesis and three rounds of selection were performed against a biotinylated inhibitor. Analysis of 84 active clones did not identify enzymes with greater activity than wild type but sequencing of the diversified region did provide insights into the mode of action of this enzyme.
An adaptation of phage display ? substrate phage, may also be used for the analysis of protease sub¬strate specificities. With this technique, the displayed moieties consist of peptides that are potential protease substrates. The peptides are sandwiched between a phage coat protein and a tag (such as c-myc) that serves to anchor the phage particle to a support. When exposed to a particular protease, only phage displaying a cleavable peptide are released into solution, whilst phage displaying non-cleavable peptides remain im¬mobilised to the support. The released phage may be retrieved and amplified and the sequencing of the inserts from recovered phage provides information about the substrate specificity of the protease used (Matthews 1996).

Phage display of antibodies
The worth of antibodies in plant research is well es¬tablished (Willats et al., 2002b). In addition to their uses for detection and isolation of cellular compo¬nents, antibodies have the unique capacity when used as immunocytochemical probes to provide contextual information at the sub-cellular level about defined epi¬tope structures (Willats et al., 1999; Willats et al. 2000). Although the number of antibodies directed against plant epitopes has grown steadily over recent decades they still cover only a minute fraction of the molecular structures involved in plant growth and de¬velopment and this shortfall is reflected by gaps in our understanding. Antibody phage display not only greatly extends our capacity to generate antibodies but also extends their potential applications for the direct functional analysis of epitopes. A further major advan¬tage of antibody production by phage display is that in many cases the whole process can be performed in vitro, thereby negating the requirement for target anti¬gens to be immunogenic. The range of feasible target antigens is therefore extended considerably because a major limitation for hybridoma antibody production is the lack of immunogenicity of potential targets. This is particularly true of glycan epitopes and is a factor that has seriously hampered antibody produc¬tion against carbohydrate plant cell wall components (Willats et al., 2000). The amount of target antigen re¬quired for antibody phage display is much less than is typically required for hybridoma antibody production (micrograms compared to milligrams) and the time re¬quired to generate monoclonal antibodies is also much reduced (a few weeks compared to several months). Because immunisation is by-passed (if single-pot li¬braries are used) the ethical and financial burdens of animal use are also avoided and phage display anti¬body production is relatively simple and cheap and requires no special facilities.
The principle of antibody phage display
Both conventional hybridoma and phage display an¬tibody production exploit the vast diversity of the mammalian antibody repertoire. The fundamental dif¬ference is that with hybridoma antibody production this diversity is harnessed by the immortilisation of antibody producing B-cells, while with phage display it is the genes that encode antibody variable regions (V-genes) that that are immortilised. (Winter et al., 1994; Marks et al., 1991; Clackson et al., 1991; Hoogenboom et al., 1998).
The principles of antibody phage display are iden¬tical to those discussed above in relation to the display of peptides and proteins. However, with antibody phage display the sequences encoding the displayed proteins are derived from genes encoding the key el¬ements of natural antibodies that determine binding. The procedures of affinity selection and screening for desired specificity are essentially the same as those de¬scribed for peptide libraries and to some extent mimic the processes of clonal selection and expansion in the mammalian immune system that underpin natural anti¬body production. Using directed evolution the binding properties of phage antibodies can be further biased towards a given target -a process analogous to affinity maturation in mammals.
Antibody phage display libraries
The mammalian V-genes that encode antibody vari¬able domains provide the raw materials for phage antibody library construction. Libraries essentially fall into two categories depending on whether these genes are derived from non-immunised animals or animals immunised with the target antigen ? single-pot and post immunisation libraries respectively (Figure 6a? c).
Post-immunisation libraries. In the construction of post-immunisation libraries, IgG sequences are gen¬erally derived from the spleen B-cells of immunised animals (Figure 6a). The repertoires of isolated V-genes are manipulated and packaged into phage li¬brary vectors. The rational is that some selection and affinity maturation of sequences with specificity for the antigen will have already occurred in vivo.Post¬immunisation libraries may therefore be pre-biased towards containing antibody fragments with desirable specificities and affinities (Hoogenboom et al., 1998). This approach has been used to create a number of valuable antibody probes against plant cell compo¬nents. Williams et al. (1996) generated a Fab fragment with specificity for the rhamnogalacturonan II (RG II) domain of the pectic cell wall matrix (Williams et al., 1996) while Shinohara et al. (2000) isolated a scFv from an post-immunisation library with specificity for the hemicellulosic fraction of Zinnia cell walls. Although high affinity antibodies can be produced using post-immunisation phage display libraries this approach has several drawbacks. Most serious is the necessity to construct a new library for every antigen so that the logistical, financial and ethical burdens of animal use associated with hybridoma antibody pro¬duction are not avoided. Moreover, because of the in vivo stage this approach requires target antigens to be immunogenic.
Single-pot libraries. Because of the limitations out¬lined above, a major goal over the past decade has been to create highly diverse, universal, antigen-unbiased libraries from which antibody fragments with specificities for a wide range of targets can be isolated. Such single-pot libraries completely avoid immunisation, library construction for all but the first user, and any immunogenic requirement. For these reasons single-pot libraries have been widely adopted and used to generate highly specific antibodies to a wide range of targets.
Two types of single-pot library have been devel¬oped ? naïve and synthetic (Figure 6b and c). In un¬immunised animals the primary, unselected antibody repertoire is dominated by IgMs with a specificity for a variety for antigens. For naïve library construction, v-gene sequences that have undergone some in vivo

Figure 6. An overview of phage display antibody library construc¬tion (a) Post-immunisation libraries are constructed using antibody gene sequences derived from animals that have been immunised with the target of interest. This approach capitalises on in vivo an¬tibody production processes, such as affinity maturation and may produce high affinity antibodies. However, a significant disadvan¬tage is that a new library must be constructed for each antigen. In contrast, single-pot libraries (b) and (c) may be used as a universal resource for the selection of antibodies against a wide range of targets. Naïve libraries (b) are constructed using V-genes sequences that have undergone some natural rearrangement, for ex¬ample sequences derived from IgM mRNA. Synthetic libraries (c) are constructed ?from scratch? using un-arranged germline V-gene sequences. (d) Antibody gene sequences are arranged and pack¬aged to produce expressed antibody fragments in various formats including single chain antibody fragments (scFvs), Fab fragments and disulphide stabilised scFvs (dsFvs).
rearrangement are derived from the IgM mRNA of an un-immunised animal (Figure 6b). This need not be an invasive process since mRNA can be sourced from peripheral blood lymphocytes (Marks et al., 1991).
Synthetic libraries are ?built? in vitro from un¬rearranged antibody gene segments with some crit¬ically positioned additional random sequences (Fig¬ure 6c). The design of synthetic libraries is based on knowledge of the key CDR (complementarity de¬termining region) sequences that shape the antigen combining site and are therefore critical for bind¬ing (Winter, 1998b; Hoogenboom and Winter, 1992). Broadly speaking the success of naïve libraries relies on their sheer size while with synthetic libraries the contents and overall diversity can be designed and controlled. Indeed, synthetic antibody libraries are be¬ing constructed that are tailor made for given epitopes (Kirkham, et al., 1999; Winter, 1998b).
Two M13-based libraries produced by the Win¬ter group at the MRC Centre for Protein Engineering (U.K.) have been widely used and have yielded high affinity scFvs to diverse targets. Both the Synthetic scFv Library (#1) and Human Synthetic VH+VL scFv Library libraries have been made available to the scientific community (Table 1). Using the Syn¬thetic scFv Library (#1) we have isolated scFvs with specificity for both protein and carbohydrate targets. Antibody PAM1 binds specifically to un-esterified ho¬mogalacturonan (HG, a component of the cell wall pectic matrix) (Willats et al., 1999) while PAM5 binds specifically to the tobacco GATA transcription fac¬tor TGAF (unpublished results). Using the Human Synthetic VH+VL scFv Library we have generated a panel of antibodies with specificity for acetylated HG. In this case phage display was successfully used after a hybridoma-based approach failed to yield anti-acetylated HG antibodies.
Antibody formats
Displayed antibody fragments can be configured in a variety of formats (Figure 6d). In the simplest arrangement, scFvs consist simply of a linear chain of natural-antibody derived heavy (VH) and light chain (VL) domains joined by additional flexible linker se¬quence. Fragments can also be designed with an engi¬neered intermolecular disulphide bond that stabilises the VH-VL pair (dsFvs). The display of antibody Fab fragments can be achieved by fusing one chain to the C-terminus of pIII and expressing the other chain un¬fused and secreted into the periplasmic space of host cells where the two chains then associate (Hoogen¬boom et al., 1998). A similar approach can be used to form bivalent and bispecific antibody fragments. Be¬cause of the small size of the inserts scFv libraries tend to be more genetically stable then Fab libraries. On the other hand scFvs are prone to dimer-and trimerisa¬tion which can hamper selection and characterisation of specificity.
Finally, the tantalising prospect of phage particles that are not based on, but mimic antibody binding has been raised by a strategy known as landscape phage display. This approach does not rely on anti¬body derived sequences at all but involves the display of thousands of copies of a peptide that cover as much as 50% of the phage surface (for example by fusion to the pVIII coat protein of filamentous fd-tet) (Petrenko and Smith, 2000). The spatial limitations imposed by the packed phage surface serve to constrain the dis¬played peptides into a defined organic surface that collectively has the capacity to bind to targets with high affinity and specificity. Since each ?landscape? varies according to the displayed peptide, a high di¬versity of specificities may potentially be generated.

Using phage display antibodies
In broad terms, phage display and hybridoma-derived natural antibodies may be used in the same range of applications, for example ELISAs, western blots and immunocytochemistry. However, phage antibod¬ies have some particular limitations and advantages. One limitation of hybridoma antibodies is that binding is usually most effective at mammalian physiologi¬cal conditions which can be a disadvantage for some plant research applications. For example, a poten¬tially powerful application of antibodies is to directly disrupt target antigens in vivo However, for many hybridoma antibodies the conditions required for bind¬ing, or even solubility, are not compatible with plant growth. With phage display it is possible to regulate screening conditions such that antibodies with binding capacity under defined conditions are isolated.
Discussed below are two aspects of phage antibody use in relation to plant science, their use as molecu¬lar probes, and for the in vivo immunomodulation of targets.

Figure 7. The use of phage display antibodies ? whole phage verses antibody fragments Both whole phage particles (a) and isolated anti¬body framents (b) may be used as immunological probes in a similar range of applications as hybridoma antibodies. The use of whole phage particles has the disadvantage that their large size leads to poor resolution. (c) In this example of immunogold labelling of tomato pericarp cell walls, the homogalacturonan epitope recog¬nised by the M13-based antibody PAM1phage is restricted to the middle lamella between adjacent cells. Arrowheads indicate the approximate extent of the middle lamella. However, PAM1phage particles are in the order of 1 µm long and covered in the pVIII coat protein that is the epitope recognised by the gold-conjugated secondary antibody. The resulting labelling therefore extends far beyond the position of the epitope since the whole length of phage particles is visualised (indicated by arrows). (d) Similarly, when PAM1phage are used for immunofluorescent microscopy their large size results in poor resolution ?fuzzy? images, as shown by this im¬age of PAM1phage binding to cells of tobacco stem parenchyma.
(e) In contrast, if the relatively small (∼ 30 KD) free PAM1scFvs are used as probes resolution is greatly increased, as shown by this image of PAM1scFv binding to cells of tobacco stem parenchyma similar to those shown in (d). PAM1scFv was detected via an N-terminal poly-histidine tag. Thanks to Dr Carolina Orfila (KVL, København) for the image shown in (c).

Figure 8. Antibody and antigen microarrays Microarrayed antibodies can be used to detect antigens, while microarrayed antigens can be used to detect antibodies. (a) Antibody microarrays are analogous to DNA microarrays in that they can be used to determine, in parallel, the relative abundance in complex mixtures of molecules isolated from two or more sources, for example mutant and wild type tissues. Differentially dye labelled extracted molecules are captured by microarrayed natural antibodies (i), antibody fragments (ii) or a mixture of antibody types (iii). The contribution of each dye to the total signal collected from a given spot is a measure of the relative abundance in the samples tested of the molecule recognised by the antibody immobilised on that spot. (b) Antigen microarrays arrays may be used to assess the binding of antibodies to immobilised antigens, for example to determine antibody specificity. Depending of the microarray platform used a wide range of antigens may be immobilised including proteins (i) carbohydrates (ii), or glycoproteins (iii). Binding to microarrayed antigens may be detected using fluorophore conjugated secondary antibodies (iv and v) or by GFP tagging or dye labelling of antibodies (vi and vii). (c) We have used antigen microarrays to assess the binding of phage display antibodies with specificity for acetylated homogalacturonan (AceHG) domains of cell wall pectic polysaccharides. Ten replicates (I-X) of ten different (1?10) AceHG samples were microarrayed onto polystyrene MaxiSorpTM treated slides (Nunc, Denmark). The differential binding of three (i?iii) different phage display monoclonal antibodies is shown. Antibody binding was detected using anti-M13pVIII /FITC secondary antibodies.

Phage antibodies as immunocytochemical probes
Phage antibody binding can be detected by the use of secondary antibodies with specificity for phage coat proteins. For example PAM1phage (Figure 7a) can be detected using secondary antibodies with specificity for the M13 pVIII coat protein. The use of anti¬pVIII secondary antibodies effectively amplifies scFv binding because of the approximately 2,700 copies of pVIII that coat the M13 phage particles (see Fig¬ure 3d). However, the large size of M13 is a disad¬vantage for immunocytochemical localisation studies because of the diffuse signal resulting from secondary antibody binding to the multiple copies of pVIII dis¬tributed along the phage particle (Figure 7c and d). In order to overcome this it is necessary to use solu¬ble (non-phage bound) scFvs. These can be produced by amplifying PAM1 in a non-suppressing host al¬though we found that this approach was inefficient. Instead, we cloned the PAM1 coding sequence from the phagemid (pHEN1, Nissim et al., 1994) into a bacterial expression vector and at the same time added a poly-histidine tag to the scFv. The soluble form of PAM1 (known as PAM1scfv, Figure 7b) can be readily isolated to high purity using a nickel resin column. PAM1phage and PAM1scfv have identical specificities although the detection limit of PAM1scfv is less than that of PAM1phage because the poly-histidine tag pro¬vides more limited binding possibilities for each sec¬ondary antibody compared to the numerous pVIII coat proteins. However, when used for immunocytochem¬ical labelling the small size of PAM1scfv provides much superior resolution compared to PAM1phage (Figure 7e).
Recently, the immunocytochemical applications of phage display antibodies have been extended by the production of scFv/GFP fusions. Functional analysis has established that in many cases such fusions can be made in which both the scFv and GFP moieties retain their original activities (Morino et al., 2001; Casey et al., 2000). Apart from providing convenient probes for quick and simple one-step immunocytochemistry, the exciting possibility is also raised of being able to express scFv/GFP fusions in planta for real time intracellular labelling of given epitopes during devel¬opmental processes. Moreover, panels of co-expressed fluophor tagged scFvs could also be used for the in vivo analysis of molecular interaction using fluores¬cence resonance energy transfer (FRET) (Truong and Ikura, 2001; Gadella et al., 1999).
Immunomodulation
Several powerful approaches are available for the dis¬ruption of plant processes and molecules at the gene level. However, if the genetic pathways controlling a particular process or molecule are not characterised an alternative strategy is to directly disrupt gene prod¬ucts in order to elucidate their functions. One such direct approach is immunomodulation -the disruption of antigen function by the action of antibody binding (Smith and Glick, 2000). This can be achieved either by micro-injection of antibodies into cells, incorpora¬tion of antibodies into plant or plant cell growth media, or by the expression of antibodies in plants. This last approach has the most practical potential for func¬tional analysis of a wide range of intracellular antigens in vivo.
One problem associated with the expression of whole antibodies or Fab fragments in plants is that the intracellular environment is not conducive to cor¬rect antibody assembly. In this regard scFvs, with their relatively undemanding folding requirements, are particularly well suited for this role and have been suc¬cessfully used to immunomodulate a variety of plant antigens (De Jaeger et al., 2000). Immunomodula¬tion of the activity of the plant hormones abscisic acid (Strauß et al., 2001) and gibberellin (Shimada et al., 1999) and the receptor protein phytochrome (Owen et al., 1992) has been demonstrated. Antibod¬ies have been targeted to the cytosol, the endoplasmic reticulum, and apoplast but in theory any cellular compartment can be targeted. However, immunomod¬ulation using scFvs is not always straightforward and many expressed scFvs are unstable. One solution is to develop more stable scFv scaffolds, another is to build antibodies free of disulphide bonds (Hoogenboom et al., 1998). Another interesting approach exploits the unusual antibodies of the Camelidae (Hamers-Casterman et al., 1993). In addition to four chain antibodies, the Camelidae produce antibodies con¬taining only heavy chains (Hamers-Casterman et al., 1993). Simple single-domain fragments (VHH) de¬rived from these heavy chain antibodies may be a valuable resource for immunomodulation (De Jaeger et al., 2000).
Microarrays for characterising and using phage display antibodies
The development of DNA microarrays has been one of the most significant bio-technological advances in recent years and is set to revolutionise the high throughput analysis of gene expression (Lander, 1999; Debouck and Goodfellow, 1999). Microarray tech¬nology is increasingly applied to the analysis of pro¬tein interactions and the analysis of antibody binding (Tomlinson and Holt, 2001; Kodadek, 2001). Arrays of antibodies (antibody arrays) can be used to detect antigens, whilst arrays of antigens (antigen arrays) can be used to detect antibodies (Haab et al., 2001).
Antibody arrays (Figure 8a) can be used for pro¬teome profiling. The rational is to isolate ligands from complex mixtures on the basis of their binding to immobilised antibodies. A typical experiment in¬volves the isolation of mixtures of proteins from say, experimental and control cells or tissue. Each pro¬tein mixture is then bulk labelled with distinguishable markers (such as Cy3 and Cy5) and exposed to the immobilised antibodies. The contribution of each dye to the total signal collected from a given spot is a measure of the relative abundance of the molecule recognised by the antibody immobilised on that spot. This parallel analysis is analogous to typical DNA mi¬croarray experiments. Antigen arrays (Figure 8b) are essentially very high throughput versions of ELISA or dot-blot assays in which the binding capacity of a particular protein ligand or antibody is assessed by its binding to a series of microarrayed potential binding partners. Binding can be detected directly if fluorophore-coupled proteins or antibodies are used or by using fluorescently labelled secondary antibodies (Figure 8b).
In our experience the time limiting step in phage antibody production is the detailed analysis of each monoclonal phage population. Conventional assays, such as ELISAs and immuno-dot-assays have the dis¬advantages that only a relatively small number of sam¬ples can be tested simultaneously, and large amounts of antibody are required for each assay. Using pro¬tein antigen microarrays, many thousands of potential binding partners can be assessed simultaneously us¬ing a very small (< 100 µl) amount of phage solution amplified simultaneously in microtitre plates. Slide surfaces coated with polylysine or super-aldehydes are available for the microarray deposition of proteins but equivalent surfaces are not available for the prepara¬tion of carbohydrate microarrays. To address this, a novel polymer microarray slide has recently been de¬veloped. The slide surface has capacity to immobilise structurally and chemically diverse glycans without any derivatisation of the slide surface or the need to create reactive groups on the glycans prior to immo¬bilisation (Willats et al., 2002a). The slides are made of polystyrene and have a surface modification known as MaxiSorpTM (NUNC A/S, Denmark) ? a surface that has been widely used in a microtitre plate for¬mat for ELISAs for many years. Using these slides we have generated carbohydrate microarrays and used them to characterise phage display antibodies with specificities for plant cell wall pectic polysaccharides (Figure 8c).

Conclusions
Phage display is a multi-purpose tool for amongst other things, molecular evolution, analysis of pro¬tein/ligand interactions and the generation of antibod¬ies. However, the relative scarcity of plant-specific examples of some applications of phage display re¬flects the fact that this technology has much still to offer plant research. As the post-genomic era pro¬gresses the emphasis of research is likely to focus increasingly on making sense of the biological con¬texts of gene products. In this respect many of the applications of phage display outlined here will be valuable tools.
Acknowledgements
Thanks to Iain Manfield, Sue Marcus, Lesley McCart¬ney, Jürgen Denecke, Carolina Orfila, Jørn Dalgaard Mikkelsen, NUNC A/S and Eva Maria Klein.

Clinical Biochemistry 35 (2002) 425?445
Review
Phage display technology: clinical applications and recent innovations
Hassan M. E. Azzazya,b,*, W. Edward Highsmith, Jra
aDepartment of Pathology, University of Maryland School of Medicine, Baltimore, USA
bDepartment of Medical & Research Technology, University of Maryland School of Medicine, Baltimore, USA

Abstract

Phage display is a molecular diversity technology that allows the presentation of large peptide and protein libraries on the surface of filamentous phage. Phage display libraries permit the selection of peptides and proteins, including antibodies, with high affinity and specificity for almost any target. A crucial advantage of this technology is the direct link that exists between the experimental phenotype and its encapsulated genotype, which allows the evolution of the selected binders into optimized molecules. Phage display facilitates engineering of antibodies with regard to their size, valency, affinity, and effector functions. The selection of antibodies and peptides from libraries displayed on the surface of filamentous phage has proven significant for routine isolation of peptides and antibodies for diagnostic and therapeutic applications. This review serves as an introduction to phage display, antibody engineering, the development of phage-displayed peptides and antibody fragments into viable diagnostic reagents, and recent trends in display technology. 2002 The Canadian Society of Clinical Chemists. All rights reserved.
Keywords: Phage display; Single-chain variable fragments; Phage-peptide; Immuno-diagnostics; Recombinant antibodies; Panning; Bispecific antibodies

Table of Contents

1. Overview and Perspectives

1.1. Introduction
1.2. Filamentous Phage
1.2.1. Structure

1.2.2. Life Cycle
1.2.3. Phagemid Cloning Vectors
1.3. The Antibody Molecule: A Synopsis
1.3.1. Structure
1.3.2. Mechanisms Responsible for Diversity of Antigen-Binding Sites
1.3.3. Antibody Fragments
2 Construction and Screening of Phage-Displayed Antibody Libraries:

2.1. Construction of scFv Phage Display Libraries
2.2. Types of Antibody Repertoires
2.2.1. Naı¨ve Repertoires: ?Single Pot Libraries?
2.2.2. Immune Repertoires

• Corresponding author. University of Maryland School of Medicine, 10 S. Pine Street, Rm 7-56, Baltimore, MD 21201. Tel.: �410-706-7011; fax: �410-706-8414.
  E-mail address: [email][email protected][/email] (H.M.E. Azzazy).
  2.2.3. Synthetic Repertoires
  2.3. Selection of Antibody Libraries: ?Bio-panning?
  2.3.1. Selection Using Immobilized Antigens
  2.3.2. Selection Using Antigens in Solution
  2.3.3. Selection on Cells
  2.3.4. In vivo Selection
  2.3.5. Remarks
  2.4. Production of Soluble scFv Molecules
  3. Improving affinity of phage antibody fragments
     3.1. Multimer Formation: Enhancing the Avidity of scFv Fragments
     3.2. Affinity Maturation by Site-Directed Mutagenesis
     3.3. Affinity Maturation by Chain Shuffling
     2 In vitro Diagnostic Applications of Phage Display

4.1. General Applications
4.1.1. Phage-Peptide Applications:
4.1.1.1. Phage display of random peptides
4.1.1.2. Mapping antibody epitopes
4.1.1.3. Generating immunogens
4.1.2. Phage-Antibody Applications:
4.1.2.1. Isolation of high-affinity Antibodies

0009-9120/02/$ ? see front matter 2002 The Canadian Society of Clinical Chemists. All rights reserved. PII: S0009-9120(02)00343-0
H.M.E. Azzazy, W.E. Highsmith Jr. / Clinical Biochemistry 35 (2002) 425?445

4.1.2.2. Catalytic antibodies
4.1.3. Directed Evolution of Proteins
4.1.4. Phage Enzymes
4.2. Production of Diagnostic Reagents for Single-Step Detection of Target Antigens: Genetic Fu¬sion of Antibody Fragments to Reporter Mole¬cules
4.2.1. ScFv-Alkaline Phosphatase Fusion Pro¬tein
4.2.2. Fluobodies
4.3. A Three-Step Strategy for Characterization of Human Sera by Phage-Displayed Peptide Librar¬ies
4.3.1. Affinity Selection of Phage-Displayed Random Peptide Libraries
4.3.2. Filter Immuno-screening of Affinity Se¬lected Phage
4.3.3. Counter-screening with Negative Sera
5. Selected Therapeutic Applications of Phage Display
5.1. Recombinant scFv Antibodies with Anti-Tumor Activities
5.2. Inhibition of Prion Propagation Using Fab Antibodies
6. Recent Innovations In Display Technology
6.1. Selectively Infective Phage (SIP)
6.2. Engineering Bispecific Antibodies
6.3. Landscape Phage Libraries
6.4. Ribosome Display
2 Conclusions
References

1. Overview and perspectives
   1.1. Introduction
   Phage display, first introduced by G. Smith in 1985 [1], is a very effective way for producing large numbers (up to 1010) of diverse peptides and proteins and isolating mole¬cules that perform specific functions (for reviews see 2?10). This technique can also be used to study protein-ligand interactions [11], receptor and antibody-binding sites [3,4], and to improve or modify the affinity of proteins for their binding partners [5,7]. Phage display involves the expres¬sion of proteins, including antibodies, or peptides on the surface of filamentous phage. DNA sequences of interest are inserted into a location in the genome of filamentous bac¬teriophage such that the encoded protein is expressed or ?displayed? on the surface of filamentous phage as a fusion product to one of the phage coat proteins (Figure 1). There¬fore, instead of having to genetically engineer proteins or peptide variants one-by-one and then express, purify, and analyze each variant, phage display libraries containing

Fig. 1. Schematic diagram of a filamentous phage displaying single chain variable fragment (scFv) molecules. The phage consists of circular ssDNA surrounded by a coat protein. g8p (pVIII) is the major coat protein whereas g3p, at the tip of the phage, is one of the minor coat proteins. The genes encoding the variable domains of the scFv and a linker are fused to gene III (g3) in the genome of the filamentous phage. Consequently, the scFv is displayed as a fusion to g3p (pIII) protein at the tip of the phage. In reality, the scFv is not fused to all g3p protein molecules, and therefore the phage retains its ability to infect bacteria. Four g3p molecules are illustrated in the figure, three of which display scFv molecules.
several billion variants can be constructed simultaneously. These libraries can then be easily used to select and purify specific phage particles bearing sequences with desired binding specificities from the nonbinding variants.
Two key discoveries were essential for the development of antibody phage display technology. First, the demonstra¬

H.M.E. Azzazy, W.E. Highsmith Jr. / Clinical Biochemistry 35 (2002) 425?445 427

tion that foreign DNA inserted into filamentous phage gene III (g3) is expressed as a fusion protein and displayed on the surface of the phage [1]. Second, the successful expression of functional antibody fragments in the periplasmic space of
E. coli [12,13].
A significant aspect of phage display lies in linking the phenotype of a bacteriophage-displayed peptide or protein with the genotype encoding that molecule, packaged within the same virion. This permits the selection and amplification of specific clones of phage representing desired binding sequences from pools of billions of phage clones. In case of filamentous phage, amplification is simply accomplished by infecting male E. coli. The genotype-phenotype linkage also permits the rapid determination of the amino acid sequence of the specific binding peptide or protein molecule by DNA sequencing of the specific insert in the phage genome.
Attempts were made to find alternative display methods such as display on bacterial surfaces [14], yeast surfaces [15], or directly on the encoding plasmid DNA (peptides¬on-plasmids library; 16). However, as all these systems still entail transformation of a cellular host, they have not suc¬ceeded in generating large diversity libraries.
This review, although not comprehensive with regard to the impressive amounts of phage display work performed over the past decade, focuses on principles of phage display technology, methods for the construction and bio-panning of phage antibody libraries, types of antibody repertoires, and different approaches to enhance the affinity of recom¬binant antibodies. It also highlights the in vitro diagnostic applications of phage-displayed peptides and antibodies, selected therapeutic applications of recombinant antibodies and recent trends in phage display technology.
1.2. Filamentous phage
1.2.1. Structure
Bacteriophages, or simply phages, are viruses that infect a variety of Gram-negative bacteria using pili as receptors. The Ff filamentous phage particles (strains M13, f1 and fd), that infect E. coli via F pili, consist of a single-stranded (ss) DNA that is enclosed in a protein coat. The entire genome of the phage consists of 11 genes. A viable phage expresses about 2700 copies of gene 8 protein (g8p or pVIII, a 50 aa residue protein that is also known as the major capsid protein) and 3 to 5 copies of the gene III (g3)-encoded adsorption protein (g3p or pIII, a 406 aa protein that is one of three minor coat proteins of the filamentous phage) on its tip (Figure 1) (for review see 17).
1.2.2. Life cycle
Filamentous phage does not produce a lytic infection in
E. coli, but rather induces a state in which the infected bacteria produce and secrete phage particles without under¬going lysis. Infection is initiated by the attachment of the phage g3p to the f pilus of a male E. coli (e.g., E. coli TG1). Only the circular phage ssDNA enters the bacterium where it is converted by the host DNA replication machinery into the double-stranded plasmid like replicative form (RF). The RF undergoes rolling circle replication to make ssDNA and also serves as a template for expression of the phage pro¬teins g3p and g8p. Phage progeny are assembled by pack¬aging of ssDNA into protein coats and extruded through the bacterial membrane into the medium.
The phage coat proteins g3p and g8p are involved in the cloning and detection of recombinant phage antibodies and peptides. Recombinant antibodies, and folded proteins, are typically expressed as g3p fusion proteins and are displayed at the tip of the M13 phage. When these antibodies bind to the antigen the bound phage is detected with an HRP-labeled antibody (Amersham Pharmacia Biotech; Uppsala, Sweden) that recognizes the g8p coat protein. Since several thousand copies of g8p exist on the phage surface, it effec¬tively amplifies the detection signal. On the other hand, peptides may be displayed as fusions to either g3p or g8p. If peptides were fused to g8p, bound phage can be detected using rat monoclonal antibodies that recognize an epitope localized in the N-terminal portion of g3p [18].
1.2.3. Phagemid cloning vectors
With the M13 phage, there are two forms of the phage DNA: ssDNA templates that can be easily prepared from phage media and used for sequencing; and dsDNA (plas¬mid-like RF) that can be isolated from the infected bacterial host and used for cloning of a target fragment. Phagemids, a more popular vector for display, are hybrids of phage and plasmid vectors. Phagemids are designed to contain the origins of replications for both the M13 phage and E. coli in addition to gene III, appropriate multiple cloning sites, and an antibiotic-resistance gene [19]. However, they lack all other structural and nonstructural gene products required for generating a complete phage. Phagemids can be grown as plasmids or alternatively packaged as recombinant M13 phage with the aid of a helper phage that contains a slightly defective origin of replication (such as M13KO7 or VCSM13) and supplies, in trans, all the structural proteins required for generating a complete phage. This process is termed ?phage rescue?. The resulting phage particles may incorporate either pIII derived from the helper phage or the polypeptide-pIII fusion protein, encoded by the phagemid. The ratios of polypeptide-pIII fusion protein: wild type pIII may range between 1:9 and 1:1000 depending on the type of phagemid, growth conditions, the nature of the polypeptide fused to pIII, and proteolytic cleavage of antibody-pIII fu¬sions.
The phagemid vector system enables coupling of affinity selection (based on the displayed repertoires of peptides or antibody fragments) to the recovery of the packaged gene encoding that peptide or antibody. Although this system imposes few limitations such as gene deletion and plasmid instability, it has been successfully used to isolate antibody fragments against a wide range of proteins, DNA, cell-surface markers, viruses and parasites. Phagemid vectors also allow either the conditional display of antibody on phage or the secretion of the antibody in the periplasmic space of E. coli in a form that can be easily detected, e.g., by ELISA.

1.3. The antibody molecule: a synopsis
1.3.1. Structure
Basic understanding of antibody structure is necessary for successful cloning of antibody genes. All antibodies have a basic structure consisting of an identical pair of heavy chain polypeptides and a pair of identical light chain polypeptides held together by disulfide bridges and nonco¬valent bonds (for a review see 20). Each of the heavy chains is encoded for by: variable (VH), diversity (D), joining (JH), and constant (CH) genetic segments; while each of the light chains is encoded for by VL, JL, and CL segments. The DNA and the amino acid sequences of the C region are relatively conserved within a given species while those of the V region are antigen-dependent. Pairing of the heavy chain V-D-J regions and light chain V-J regions creates an anti¬gen-binding site (paratope) which recognizes a single anti¬genic determinant (epitope). Each V region consists of an alternating framework (FW), which is more conserved, and three hyper-variable or complementarity-determining re¬gions (CDRs) with greatest sequence diversity. The CDRs, and to a lesser extent the FW regions, interact with the antigen to form the core of an antigen-binding site. The first 2 CDRs are encoded by the V segment while the third CDR is the product of the junction of V-D-J for the heavy chain or V-J for the light chain. This knowledge of antibody structure has facilitated the creation of antibody molecules entirely outside their natural host.
1.3.2. Mechanisms responsible for diversity of antigen-binding sites
An enormous number of different antibodies can be generated by the immune system, each having a unique antigen-binding site (paratope) generated by the N-terminal domains of the heavy and light chains. Several mechanisms are responsible for the diversity of paratopes [21]: (i) a recombinatorial diversity: created by random selection of one variable heavy chain gene (VH), one diversity gene (D) and one heavy joining (JH) minigene, or one variable light chain gene (VL) and one light joining (JL) gene segment out of a pool, to constitute the VH and VL domains, respec¬tively; (ii) a junctional diversity: appended by the imprecise joining mechanisms and by deletion or addition of random nucleotides at the borders of the recombining VH-D-JH minigenes; and (iii) a combinatorial diversity: generated by the assembly of the VH and VL domains. Other mechanisms include: (i) enlargement of the architecture of the paratope by adjusting the angle between the associated VH and VL domains; and (ii) specific maturation of the paratope caused by a somatic hypermutation that improves the shape complementarity of the antibody with the antigen. The cu-
Table 1 Characteristics of Antibody Fragments
Antibody Size Paratopes Structure Fragment (kDa) (Valency)
ScFv 25?30 1 VHand VL domains are linked by a 15 aa linker. Changing the linker length directs the formation of diabodies (60 kDa), triabodies (90 kDa) or tetrabodies (120 kDa)
Fv 25 1 VH and VL with no linker between the V-domains
Minibody 80 2 An scFv-CH3 fusion protein that self-assembles into a bivalent dimer of 80 kDa
Fab 50 1 Composed of two chains:VH-CH1 and VL-CL
F(ab�)2 100 2 Two Fab molecules
IgG 150 2 Parent antibody molecule: two heavy chains (VH-CH1-CH2-CH3) and two light chains (VL-CL)
mulative effects of these mechanisms determine the anti¬genic affinity and specificity of an antibody.
1.3.3. Antibody fragments
Antibody molecules contain discrete protein domains that can be separated by protease digestion or produced by recombinant technology. Two antigen-binding fragments designated Fab and Fv have been cloned and displayed on phage. The larger Fab (fragment antibody) consists of VH-CH and VL-CL segments linked by disulfide bonds. The smaller Fv (fragment variable) is composed of the VL and VH regions only. The recombinant version of the Fv is termed the single-chain variable fragment (scFv). The two variable regions in the scFv are artificially joined with a flexible peptide linker, usually a 15 aa linker is used with the sequence (Gly4Ser)3, and expressed as a single polypeptide chain. The linker allows the association of the VH and VL to form the antigen-binding site. A summary of the character¬istics of different antibody fragments is presented in Table

1. Schematic representations of intact immunoglobulin mol¬ecule and several monomeric antibody fragments are pre¬sented in Figure 2.
2. Construction and screening of phage-displayed antibody libraries
   2.1. Construction of scFv phage display libraries
   One of the most powerful applications of phage display has been the isolation of recombinant antibodies with a unique specificity [3,4,7,8,22]. In this regard, phage display technology can be used to: (i) generate human monoclonal antibodies or humanize mouse antibodies, significant for cancer immunotherapy, (ii) isolate human antibodies from patients exposed to certain viral pathogens to better under¬

H.M.E. Azzazy, W.E. Highsmith Jr. / Clinical Biochemistry 35 (2002) 425?445 429

Fig. 2. Structure of intact immunoglobulin G (IgG) molecule, a minibody, and monomeric antibody fragments: Fab (antibody fragment), Fv (variable fragment), and scFv (single chain variable fragment). Variable domains of heavy (VH) and light (VL) chains are represented by black and white ovals, respectively. Constant regions of heavy (CH1-3) and light (CL) chains are represented by shaded and dotted ovals, respectively. ABD � antigen-binding domain (paratope).

stand the immune response during infection and how pro¬tective antibodies are generated, and (iii) elucidate the spec¬ificity of autoimmune antibodies. Both Fab and scFv were expressed on the surface of M13 viral particles with no apparent loss of the antibody?s specificity and affinity.
To construct an scFv library using phage display, genes of variable heavy (VH) and variable light (VL) chains of antibodies are prepared by reverse transcription of mRNA obtained from B-lymphocytes. The heavy and light chain gene products are amplified and assembled into a single gene using a DNA linker fragment. The assembled scFv DNA fragment is inserted into a phagemid vector and the recombinant phagemid is introduced into competent E. coli by CaCl2 transformation or electroporation. Ligation and bacterial transformation are crucial, as they directly influ¬ence the size of the library. Phagemid-containing bacterial cells are grown and then infected with a helper phage (M13VCS or KO7) to yield recombinant phages that display scFv antibody fragments as fusion to one of the phage coat proteins. A detailed description of the steps involved in the construction of an immune scFv repertoire using phage display is shown in Figure 3. A summary of different vari¬ables considered for constructing an antibody library is presented in Table 2.
The size of antibody repertoires has been limited to �108 clones by the efficiency of DNA transfection into bacterial cells (as described above). Larger antibody repertoires of
�1010 clones have been prepared [23] using the process of combinatorial infection and in vivo recombination [24]. In this method, bacteria containing a repertoire of ?donor� heavy chains (encoded on a plasmid replicon) are infected with an ?acceptor? light chain repertoire (on phage). The two chains are then recombined on the same phage replicon within the bacterium. Using this process, antibody frag¬ments against a range of haptens and antigens, with affini¬ties in the nanomolar range, have been obtained.
The quality of the library is important for its perfor¬mance. Library quality control can be achieved by checking the following parameters: (i) number of clones whose pha¬gemids contain the scFv insert, (ii) number of clones ex¬pressing phages carrying scFv, and (iii) number of clones expressing soluble scFv. These parameters can be assessed by a variety of methods including PCR screening of indi¬vidual clones (to detect the presence of the scFV insert), and

H.M.E. Azzazy, W.E. Highsmith Jr. / Clinical Biochemistry 35 (2002) 425?445

dot blot analysis (to detect both phages displaying scFv and soluble scFv produced by the screened clones). Positive clones can be further characterized by DNA fingerprinting, using a frequent cutter restriction enzyme, of PCR-ampli¬fied scFv inserts and DNA sequencing.
2.2. Types of antibody repertoires
2.2.1. Naı¨ve repertoires: ?single pot libraries?
In this strategy, V-genes from the IgM mRNA of B-cells of unimmunized human donors are isolated from peripheral blood lymphocytes, bone marrow, spleen cells, or from animal sources. From small-sized human single-pot librar¬ies (3 � 107 antibody clones), antibodies have been isolated against ?foreign? antigens (e.g., bovine serum albumin and lysozyme), haptens (2-phenyloxazol-5-one), or ?self? anti¬gens (thyroglobulin, tumor necrosis factor �) [25]. The affinity of these antibodies was similar to that seen in na¨ıve primary immune response and was sufficiently reactive in Western blot, ELISA, and FACS analysis [25].
A large sized na¨ıve library was constructed using lym¬phocytes from over 40 nonimmunized human donors and contained 1.4 � 1010 clones [26]. Antibodies were isolated from this library against all antigens tested with affinities (Ka) in the low nanomolar range, typical for a secondary immune response [26]. Therefore, neither immunization nor affinity maturation are required for generating human anti¬bodies with high affinity.
Key advantages of single-pot repertoires include: (i) iso¬lation of human antibodies to self, nonimmunogenic or toxic antigens; (ii) a single library can be used for all antigens; (iii) short time needed for antibody generation (2?4 rounds of selection in two weeks); and (IV) direct isolation of high affinity antibodies when very large reper¬toires are used. Disadvantages of na¨ıve libraries are: (i) low affinity of antibodies isolated from small sized libraries; (ii) the time needed to construct large libraries, and (iii) content and quality of the library are influenced by the unequal
Fig. 3. Schematic diagram for constructing an immune scFv repertoire using phage display. Total RNA is first isolated from spleen cells of B-lymphocytes and then mRNA is affinity purified by affinity chromatog¬raphy on oligo(dT)-cellulose. mRNA is reverse transcribed into cDNA using random hexamers. The heavy and light chain antibody genes are amplified in two separate reactions using two sets of primers designed to hybridize to opposite ends of the variable region of each chain. The purified heavy and light chain DNA products are assembled into a single gene using a DNA linker fragment constructed to hybridize to the 3�-end of the heavy chain and the 5�-end of the light chain. The assembled scFv DNA fragment is amplified by PCR using a set of primers designed to introduce restriction sites for cloning into a phagemid vector. Following restriction digestion and ligation of the scFv DNA to the phagemid, the ligated vector is introduced into competent E. coli by CaCl2 transformation or electropora¬tion. Phagemid-containing bacterial cells are grown and then infected with a helper phage (M13VCS or KO7), a process known as phage rescue, to yield recombinant phages which display scFv antibody fragments as fusion to one of the phage coat proteins.

H.M.E. Azzazy, W.E. Highsmith Jr. / Clinical Biochemistry 35 (2002) 425?445 431

Table 2 Variables Considered in Making an Antibody Library
Variable Remarks

Source of variable domain genes and CDRs: High affinity antibodies to immunogenic or disease-related antigen can be isolated from a) Immune or disease-related library immune or disease libraries b) Natural or synthetic libraries Natural or synthetic libraries are better source for antibodies to many antigens

Amplification of antibody genes by: ? a) PCR b) Synthetic oligonucleotides c) Combination

Format of the displayed fragment: ScFv molecules have higher potential to form multimers a) scFv Size (scFv:25 kDa, Fab 50 kDa) b) Fab Stability issues (Fab is more stable)
Display system: ● Display in many copies using the major coat protein g8p (pVIII), or in few copies a) Phage or phagemid display using the g3p using the minor coat protein g3p (pIII) (pIII) or g8p (pVIII) ● For multivalent display using the g8p (pVIII), it will be more difficult to select
higher affinity clones
b) Cellular display systems: E. coli or yeast ● Phagemid systems using g3p (pIII) display are the most frequently used as they allow high frequency of monovalent display and easy conversion from phage-displayed antibody to soluble antibody
c) Ribosome display ● For cellular display systems, cell sorting may be used to enrich affinity variants
● Ribosome display offers a cell-free molecular evolution system for antibodies

expression of the V-genes repertoire, unknown history of the B-cell donor, and potential limited diversity of the IgM repertoire.
2.2.2. Immune repertoire
In this library, V-genes are derived from the IgG mRNA of B-cells from an immunized animal or, in certain cases, human. Humanized immune repertoires can also be pre¬pared using immunized transgenic mice (xenomice). Xe¬nomice have been generated that harbor most of the human immune system V-genes in the germline (instead of the native murine immune repertoire) [27]. Immunization of these mice with a hapten or a foreign antigen results in the production of human-like antibodies in their B-cells. The antibody genes can be recovered from the B-cells by PCR and library selection or by fusion into a monoclonal cell line by hybridoma technology.
An immune antibody library has two main character¬istics: (i) it will be enriched in antigen-specific antibod¬ies, and (ii) some of these antibodies will have undergone affinity maturation by the immune system [13,28]. Im¬mune libraries were used to produce antibodies against carcino-embryonic antigen (CEA) [29], T-cell recep¬tor-V� [30] and major histocompatibility complex/pep¬tide complexes [31]. High-affinity antibodies were re¬portedly derived from mice [31], chickens [32], and rabbits [33]. Antibody libraries were also derived from sheep [34], cows and nonhuman primates [35].
Disadvantages of immune libraries include: (i) long time required for animal immunization, (ii) lack of im¬mune response to self or toxic antigens, (iii) the unpre¬dictability of the immune response to the antigen of interest, (iv) a new antibody library must be constructed for each antigen (this increases the total time of the procedure by 1?3 months), and (v) restrictions in gen¬erating human antibodies.
A unique application of immune libraries is to clone high-affinity antibodies present after viral infections or can¬cer, and antibodies to self-antigens present in patients with autoimmune diseases. Analysis of such antibodies could aid in the identification of antigenic epitopes involved in the humoral immune response. A second application of immune libraries involves the removal of irrelevant antibodies; this can be used to generate antibodies against minor or poorly immunogenic antigens. Animals can be made tolerant to certain antigens then immunized with a mixture of the relevant antigen and the irrelevant ones. This approach was used to isolate human antimelanoma antibodies from phage libraries of cancer patients immunized with autologous tu¬mor cells [36].
2.2.3. Synthetic repertoires
The specificity of any antibody resides in the six complementarity determining regions (CDRs) that shape the antigen binding sites (3 CDRs on each of the variable heavy and variable light chains). It became obvious from structural studies that five of the six CDRs have limited structural variation [37]. The CDR3 of the heavy chain (VH-CDR3) is the most diverse loop in composition and length (estimated potential diversity of 1023 sequences)
[38] and is most central to the antigen-binding site of all CDRs. Therefore, synthetic repertoires can be made by randomizing the VH-CDR3 region using oligonucleotide-directed mutagenesis or PCR-based techniques. Several synthetic repertoires were made using different strategies including the use of randomized light and heavy chain CDR3s [39] and diversifying all three CDR loops in one V-gene segment [40]. A major advantage of synthetic repertoires over na¨ıve ones is the potential to control and define the contents, local variability and overall diversity of synthetic libraries.

2.3. Selection of antibody libraries: ?bio-panning?
Antibody libraries are screened and enriched for antigen-specific clones by a technique known as bio-panning in which phages displaying scFv are incubated with an immo¬bilized antigen of interest [25,28]. Unbound phages are removed by washing whereas phages displaying scFv that specifically bind the antigen are eluted, by changing the binding conditions, and amplified in E. coli. This selection cycle is illustrated in Figure 4. Ideally, only one cycle of selection should be required, however the binding of non¬specific phage limits the enrichment that can be achieved per cycle. In practice, several rounds of selection are nec¬essary (average 2?4 cycles). Several bio-panning strate¬gies (Figure 5) are discussed below.
2.3.1. Selection using immobilized antigens
Phage libraries are selected by flowing through an affin¬ity column with the immobilized antigen of interest [28,41]. Following washing of the column to remove nonspecific clones, specific binders are eluted and amplified in E. coli. Selection can also be performed against antigen adsorbed onto plastic surfaces such as immunotubes (Maxisorb tubes; Nalge Nunc Intl., Naperville, IL) or enzyme-linked immu¬nosorbent assay (ELISA) plates [42,43]. Alternatively, an¬tigen may be immobilized on chips of BIAcore sensors [44].
It should be noted that selection of the immobilization method must take into consideration the conformational integrity of the immobilized antigen. Some phage antibod¬ies selected against an adsorbed antigen may not be able to recognize the native form of the antigen. One way to cir¬cumvent such problem is to employ indirect antigen coating through the use of antigen-specific capture antibodies [45].
Specific phage-displayed antibodies can be eluted from their specific antigens with acidic solutions (such as HCl or glycine buffer) [43,46], with basic solutions such as trieth¬ylamine [42], by enzymatic cleavage of a protease site constructed between the antibody and g3p [47], or by com¬petition with excess antigen [28].
2.3.2. Selection using antigens in solution
This technique allows solution binding and overcomes issues with conformational changes that are encountered upon coating antigens on solid surfaces. The use of labeled soluble antigens also allows a more accurate quantification of the antigen used during selection [48] and consequently enhances the ability to use lower concentrations of the antigen to favor selection of high-affinity phage antibodies. Following incubation of phage-antibodies with biotinylated antigen, phage bound to the labeled antigen are recovered with avidin or streptavidin-coated paramagnetic beads. Spe¬cific phages are then dissociated from the antigen and char¬acterized. One disadvantages of this technique is that anti¬streptavidin antibodies will also be isolated. However, this problem can be resolved by a depletion step using strepta¬vidin-coated beads.
2.3.3. Selection on cells
Direct selection of antibodies against markers on cell surfaces may be carried out on either monolayers of adher¬ent cells or on cells in suspension. Unbound phage can be washed away by rinsing tissue culture flasks (monolayers) or centrifugation (cell suspension). To optimize the isola¬tion of antigen-specific binders and minimize the binding of irrelevant binders, a simultaneous positive and negative selection may be applied [49]. In this approach, a compe¬tition is set up between a small number of antigen-positive target cells and an excess of antigen-negative ?absorber? cells to bind antibodies of phage library; the absorber cells serve as a sink for the nonspecific adherence of irrelevant binders. A fluorescently labeled antibody against an irrele¬vant antigen present only on the target cells is added and FACS is used to isolate the target cells binding the specific phage antibodies. Using a similar technique and a Fab library researchers isolated several anti-Rh(D) Fab antibod¬ies of clinical significance [50]. Similar approaches can be utilized to identify putative tumor-specific antigens and pro¬vide a quick high-yield approach for isolating self-replica¬tive antibody fragments directed against novel or conforma¬tionally dependent cell surface markers. Another group subjected a scFv library to three rounds of positive selection on human melanoma cells and negative selection on human peripheral blood mononuclear cells [51]. Two scFv clones were isolated that recognize melanoma cells in ELISA and FACS [51]. Selections may also be carried out on tissue sections as well as whole tissues.
2.3.4. In vivo selection
In this method phage repertoires are directly injected into animals and then tissues are collected and examined for phage bound to tissue-specific endothelial cell markers as was demonstrated for peptide phage [52]. Pasqualini and Ruoslahti [52] were the first to isolate phage-displayed peptides that home to selective vascular beds in vivo. In vivo panning has several advantages: (i) the isolated phage-dis¬played peptides home selectively to ?intact? targets of in¬terest; (ii) an inherent blocking step is included where most of the phage-displayed peptides that recognize ubiquitous plasma and cell surface proteins are eliminated; (iii) these peptides may be useful for the functional analysis of new receptors and potential identification of novel drug target candidates because some of the isolated peptides have been found to bind to endothelial receptors expressed in the vasculature of specific tissues.
2.3.5. Remarks
Many laboratories have experienced practical difficulties with screening phage libraries. For example, panning of

H.M.E. Azzazy, W.E. Highsmith Jr. / Clinical Biochemistry 35 (2002) 425?445 433

Fig. 4. Bio-panning of phage-display library. The library is screened in four steps: (1) binding of phages to the target antigen (in this case antigen immobilized on a solid support), (2) washing to remove unbound phage, (3) dissociation to recover antigen-specific phage, and (4) amplification of the antigen-specific phage by infection of E. coli. Caution must be exercised during the binding and washing steps to avoid low-ionic strength or other conditions that may favor adsorption of phage directly onto plastic or other matrix.

Fig. 5. Different approaches for panning of phage libraries. Antigen col¬umns: phage libraries can be selected by flowing the library through a column with immobilized antigen. Coated antigens: phage libraries can be selected on antigen adsorbed on plastic surface. Selection using biotinyl¬ated antigens in solution: this method is used to evade conformational change of antigen that occurs during coating. Bound and unbound phages are separated using avidin-coated magnetic beads. Selection on cells: is performed directly on monolayers of cells or on cells in suspension; unbound phages are removed by gentle rinsing or centrifugation. In vivo selection: in this method phage libraries are directly injected into animals and then tissues are collected and examined for phage bound to tissue-specific antigens.
antibody libraries suffers from failing to enrich for specific antibodies and recovery of only low-affinity antibodies. Selective loss of high-affinity phages during the selection cycles may be corrected by increasing the stringency of the phage elution from the target antigen. On the other hand, monitoring the antibody gene insert size in phagemids be¬tween two panning cycles indicated that the fraction of phages with full-size insert decreased during amplification [53]. Phages with smaller insert (usually missing VH region) tend to overgrow phages with full-size inserts in mixed cultures. This may be abolished by picking a number of individual phagemid clones (with full-size inserts) after the last panning cycle and mix pure cultures for a ?polyclonal? antibody. Therefore, adjusting the elution and screening procedures during selection can determine whether high-affinity phages are selected or discarded.
2.4. Production of soluble scFv molecules
To produce soluble scFv, antigen-positive phages are used to infect a specific strain of E. coli (e.g., E. coli HB2151) that will direct production of soluble scFv. Such
E. coli strain is termed ?nonsuppressor? cells as they rec¬ognize an amber stop codon, engineered between the scFv gene and g3 in the phagemid, and only express the scFv without the g3p protein. The phagemid is also designed to introduce polyhistidine tag fused to the expressed scFv, thereby permitting rapid and simple protein purification by immobilized metal affinity chromatography. Depending on the isolated clone, soluble antibodies may be present in the culture supernatant, the bacterial periplasm, and/or inside the bacterial cells. All three fractions must be isolated and analyzed in a Western blot, using a commercially available conjugated antihistidine (His) tag antibody, to determine the location of the soluble antibodies. Soluble scFv are rela¬tively simple to isolate, can be economically produced in bacteria in very large quantities [54,55], and do not entail complex refolding procedures [56].
3. Improving affinity of phage antibody fragments
Although the antibodies selected from many immune or even large sized single-pot libraries may be extremely use¬ful for research purposes in ELISA, Western blots, or im¬munofluorescence; their affinity is often not sufficient for use as sensitive diagnostic reagents.
Affinity maturation may be circumvented by making multivalent molecules [57]; however, in vitro affinity mat¬uration of selected antibodies may still be required in certain instances. V-gene chain shuffling [28] and site-directed mu¬tagenesis are used for affinity maturation of scFv molecules. Other methods for randomization of protein sequences were also applied including propagation of the scFv genes in E. coli mutator strains [58], error-prone PCR [48], and DNA shuffling [59].
3.1. Multimer formation: enhancing the avidity of scFv fragments
The scFv molecule is about 30 kDa in size and consists ofaVH and VL domains tethered together via a polypeptide linker (usually 15 amino acid residues) that serves to im¬prove expression and folding efficiency. A scFv molecule is

H.M.E. Azzazy, W.E. Highsmith Jr. / Clinical Biochemistry 35 (2002) 425?445 435

monovalent with a single functional paratope; consequently it has low avidity. Using a short linker of 5 amino acid residues between the VH and VL domains of the scFv was found to prevent the alignment of V-domains into a single scFv and lead to the association of two scFv molecules forming a dimer termed a ?diabody? [60,61]. A diabody, with a molecular size of about 60 kDa, is an scFv dimer with two functional paratopes and higher avidity than scFv frag¬ments. Reducing the length of the linker to three residues prevented the formation of scFv dimers and instead directed the association of three scFv molecules to form a trimer (a 90-kDa triabody with three paratopes) [61] and/or a tetrava¬lent tetrabody [62]. Bispecificity can be also achieved by fusing two different scFv [63,64]. Clearly, this approach permits the design of multi-specific binding molecules with chosen size and functional affinity.
3.2. Affinity maturation by site-directed mutagenesis
In this method, the amino acids of one or more of the CDRs are substituted with different residues followed by the selection of clones with higher affinity for the target antigen. A refinement of this method ?parsimonious mu¬tagenesis? was developed [65]. In this strategy, the entire CDR sequence of an antibody fragment is screened to iden¬tify amino acids that are actively involved in the antigen binding. First, the number of codons introduced is limited such that each amino acid is only coded for by a single codon. The amino acids at each position are then manipu¬lated to favor parental sequences, conservative changes, and those that appear more frequently in CDR regions of the antibody. Because parsimonious mutagenesis provides in¬formation about the contribution of distinct amino acids to binding and affinity, this process can be used as an initial step in the affinity maturation of recombinant antibodies.
3.3. Affinity maturation by chain shuffling
Chain shuffling provides alterations to the intrinsic af¬finity of a specific monovalent scFv. In this process, the scFv molecule is subjected to a series of manipulations in which the gene for one chain (e.g., VH) of the scFv molecule is cloned into a repertoire for the second chain (VL) [66]. The resulting scFv library will contain the scFv-phage with VH chains specific for the target antigen and random VL chains. The library is panned against the antigen to identify clones with improved binding properties. The new VL gene is then cloned into a repertoire of VH genes. To maintain the specificity of the parent scFv molecule, the VH CDR3 (that contains most of the contact residues to the antigen) is retained without changes. Therefore, only the VH segment from frameworks 1 to 3, including the CDRs 1 and 2, are replaced. Using this strategy, the affinity of a nonimmune human scFv which binds the glycoprotein tumor antigen c-erbB-2 (Kd � 1.6 � 10�8 M), was increased sixfold (Kd � 2.5 � 10�9 M) by light-chain shuffling and fivefold (Kd
Table 3 Example Peptide Ligands Selected from Phage Display Libraries
Target Protein Peptide Sequence Reference
Atrial natriuretic peptide *MCHFGGRMDRISCYR 155
receptor-A
Concanavalin A MYWYPY 75
GPIIb/IIIa ?CNWKRGDC 156
Steptavidin AECHPQGPPCIEGRK 157
Thrombin receptor MSRPACPNDKYE 79
TPO (human thrombopoietin IEGPTLRQWLAARA 158
receptor)

• A disulfide bond between underlined cysteine residues leads to the formation of a cyclic peptide
  ? Both the C-and N-termini of this peptide are modified as �CONH2 and �OCCH3, respectively.
  � 3.1 � 10�9 M) by heavy-chain shuffling, values compa¬rable to those for antibodies against the same antigen pro¬duced by hybridomas [67].

4. In vitro diagnostic applications of phage display
   4.1. General applications
   4.1.1. Phage-peptide applications
   4.1.1.1. Phage display of random peptides. Synthetic oligo¬nucleotides with a constant length but with unspecified codons, randomized through site-directed mutagenesis us¬ing degenerate oligodeoxynucleotides, are cloned as fusions to one of the coat proteins of M13 phage where they are expressed as peptide-capsid fusion proteins. Phage-borne peptides exhibit a wide mimicking potential to linear, con¬formational and nonproteinaceous epitopes [68,69]. These random peptide libraries can be tested for binding to target molecules of interest (Table 3). The display of random peptides on filamentous bacteriophage as fusion to either g3p or g8p coat proteins [1,70 ?72] has allowed the identi¬fication of peptides that specifically bind to a variety of targets including antibodies [73], streptavidin [74], ribonu¬clease S [69], and concanavalin A [75].
   Peptides selected against a particular target that had similar sequences played a role in identifying a sequence motif necessary for binding [76]. In cases where the selected peptides did not resemble the natural peptide ligand, they were termed mimotopes [77,78]. Peptide sequences identi¬fied by phage display have been shown to act as agonists and antagonists of receptors [79]. Peptides that neutralize immunoglobulins may be employed as diagnostic reagents (see Section 4.3.) or used as therapeutic agents for control¬ling autoimmune diseases [80]. Random peptide libraries can be used for mapping epitopes of monoclonal and poly-clonal antibodies, identifying peptide ligands, and develop¬ing ?substrate phage? to define substrate sites for different enzymes [81,82]. Finally, display of small peptides on the surface of phage particles can increase their immunogenic¬ity and consequently their potential as vaccine candidates.

4.1.1.2. Mapping antibody epitopes. Fragments of DNA that encode parts of the protein antigen are fused to a gene encoding one of the capsid proteins. Phage particles dis¬playing antigenic peptides can be used for mapping epitopes of monoclonal and polyclonal antibodies [83]. Phage dis¬play libraries of random peptides have also proven useful for identifying antibody epitopes in cases in which the antigen is not available or even not yet known [84].
4.1.1.3. Generating immunogens. Small segments of differ¬ent proteins displayed on M13 virus particles were used to elicit antibodies against the coat proteins of parasites and viruses. The immunologic response to injected M13 phage is T-cell dependent and does not require adjuvant.
4.1.2. Phage-antibody applications
4.1.2.1. Isolation of high-affinity antibodies Phage-dis¬played recombinant antibodies have several advantages over monoclonal antibodies generated by hybridoma tech¬nology. In comparison to the time-consuming and labor-intensive cell screening processes of hybridoma production, antibody genes can be cloned directly from spleen cells using rapid recombinant DNA methods. Generation of a large natural display library from variable gene repertoires can eliminate animal immunization and large-scale cell cul¬ture for hybridoma development and allow isolation of antibodies with high affinity against any antigen. Phage display is particularly useful in cases where monoclonal antibodies could not be obtained by classical hybridoma technique such as antibodies against nonimmunogenic or toxic antigens. Phage displayed antibodies have stable genetic source. Phage antibody technology can also be used to clone and rescue monoclonal antibodies from genetically unstable hybridomas. Phage antibody genes can be easily sequenced, mutated, and screened to im¬prove antigen binding. Finally, soluble recombinant an¬tibodies (not displayed on phage) can be produced quickly and economically and can be used as in vitro diagnostic reagents.
4.1.2.2. Catalytic antibodies Catalytic antibodies are gener¬ated by immunization with haptens that are analogs of rate-determining transition state structures in a particular chemical reaction. The energy associated with the formation of the antibody-transition state complex decreases the acti¬vation energy for the chemical reaction and increases the reaction rate.
The ability of the native immune system has already been exploited to obtain catalytic antibodies [85]. Antibody phage libraries should also prove a useful source of catalytic antibodies. A potentail added advantage for searching anti¬body libraries for catalytic antibodies is the ability to select for actual catalysis rather than for binding activity only [86].
4.1.3. Directed evolution of proteins
The coding region of a protein, that possesses useful physicochemical or other properties, can be altered by cas¬sette mutagenesis, error-prone PCR or shuffling to generate a group of modified sequences. A library of recombinant phages displaying a multitude of variants of the parental protein is constructed and affinity selected to isolate fusion phages that display variants with the highest binding char¬acteristics for a target. Because each phage particle links a particular gene (located inside the particle) to the gene-encoded protein displayed on the phage surface, the primary structures of the desired protein variants can be elucidated by DNA sequencing. This approach was used to identify novel enzyme inhibitors and antagonists [87]. Most recently a novel database has been established that incorporates data on full-length proteins, protein domains and peptides that were obtained through in vitro directed evolution processes mainly by phage display [88].
4.1.4. Phage enzymes
Several enzymes have been displayed on M13 bacterio¬phages and retained their catalytic activities. These include alkaline phosphatase [89], trypsin [90], and �-lactamase [91]. In theory, any enzyme that can be expressed in E. coli may also be displayed on M13 phage.
Phage display libraries based on suitable enzymes can improve diagnostics by enhancing the stability and catalytic activities of enzymes, and probably enabling the engineer¬ing of catalysis that is modifiable by antigen binding.
4.2. Production of diagnostic reagents for single-step detection of target antigens: genetic fusions of antibody fragments to reporter molecules
The antibody-fusion proteins provide high sensitivity in diagnostic assays because they enable signal amplification after the initial binding of the antibody to its target antigen. Antibody fragments have been fused to alkaline phospha¬tase, green fluorescent proteins, and lipids [92] for diagnos¬tic applications. In practice, recombinant designs for anti¬body fusions are only limited by an expression system capable of producing correctly folded protein products.
4.2.1. ScFv-alkaline phosphatase fusion protein
Harper et al. subcloned the DNA encoding the antiluteo¬virus scFv, obtained from a synthetic phage antibody library after four rounds of panning, into the expression vector pDAP2 [93]. Following transformation of the recombinant plasmid into bacteria and induction with IPTG (isopropyl¬
�-D-thiogalactopyranoside, an inducer of the lac operon), a bifunctional fusion protein consisting of scFv fused to the N-terminus of alkaline phosphatase was produced. An oli¬gonucleotide sequence encoding hexa-histidine was added to the 3� end of the alkaline phosphatase gene to allow purification of the fusion protein by immobilized metal affinity chromatography. Because alkaline phosphatase is a

H.M.E. Azzazy, W.E. Highsmith Jr. / Clinical Biochemistry 35 (2002) 425?445 437

dimer, an additional advantage of its fusion to scFv is the simultaneous production of a bivalent antiluteovirus scFv that should have a greater avidity than monomer scFv [94]. The scFv-alkaline phosphatase fusion protein was used di¬rectly in ELISA to detect luteovirus in sap extracts from infected plants. These results demonstrate the feasibility of fusing scFv to alkaline phosphatase for direct single-step detection of target antigens.
4.2.2. Fluobodies
Antibodies conjugated to the fluorochrome fluorescein isothiocyanate (FITC) have been used extensively in immu¬nofluorescence and phenotyping by flow cytometry tech¬niques for the diagnosis of various diseases [95]. However, FITC is sensitive to photo-bleaching by illumination. Green fluorescent proteins (GFP) were reported to have compara¬tive fluorochrome characteristics to FITC, however they have increased stability. Griep et al. [96] developed an expression system (pSKGFP) which permits the expression of scFv as fusions with GFP ?fluobodies?. A His-tag was added to the C-terminus of the GFP to allow purification of fluobodies by immobilized metal affinity chromatography. Fluobodies against lipopolysaccharides of the bacterium Ralstonia solanacearum were produced in bacterial cultures and shown to function well in flow cytometry and immu¬nofluorescence cell staining. Fluobodies retain their speci¬ficity for their target antigens and, unlike FITC-conjugated antibodies, they do not fade upon illumination. Further¬more, the investigation of fluobodies with other spectral properties might allow simultaneous multiple labeling of different epitopes.
4.3. A three-step strategy for characterization of human sera by phage-displayed random peptide libraries
The onset of a disease is frequently associated with the appearance in patient?s serum of specific antibodies that target all of the infectious agent determinants that are im¬munologically relevant to the host. The detection and char¬acterization of such antibodies could lead to the discovery of disease-specific epitopes and may have great diagnostic and prognostic significance. Also, the identification and staging of several infectious diseases depend mainly on the characterization of a specific immune response in the pa¬tient. Such analysis requires identification of the natural antigen against which the immune response is directed. This may be hampered if the antigen is unavailable or is simply unknown.
Extensive evidence has accumulated to substantiate the conclusion that phage displayed libraries of random pep¬tides have specific ligands for almost any given antibody [97]. A three-step strategy was developed [98] to identify disease-specific phage-borne peptides that entails: (a) affin¬ity selection of peptide libraries, (b) screening of selected phage by sera from other patients, and (c) counter-screening with negative sera. The entire procedure can be accom¬plished in absence of information about the etiologic agent or its antigens.
4.3.1. Affinity selection of phage-displayed peptide libraries
A phage peptide library is affinity-selected with one disease-specific serum. Affinity selection can be accom¬plished by direct coating of partially purified immunoglobu¬lins onto the surface of polystyrene beads, or tethering immunoglobulins on the surface of magnetic beads via the use of antihuman Fc secondary antibodies covalently linked to the beads. The later method has two advantages: (i) proper orientation of the serum immunoglobulins that al¬lows the maximum number of available antigen-binding sites to interact with the target peptides, and (ii) it avoids the partial denaturation of serum antibodies that may result from their direct coating on the beads.
4.3.2. Filter immuno-screening of affinity-selected phage
In conventional selection of peptide libraries using puri¬fied antigens, several rounds of selection are often needed to eliminate nonspecific clones. However, in the growth cycle that follows each round of selection, different phages can be amplified at different rates thus introducing a biologic se¬lection that does not relate to that obtained by affinity. Furthermore, because polyclonal antibodies have a wide spectrum of affinities and concentrations; several rounds of selection may lead to the amplification of peptide-phages that are recognized by only particular antibody subsets. Because no information is available on the affinity and/or the concentration of disease-specific antibodies, it becomes very difficult to predict whether multiple selection rounds would effectively favor enrichment of disease-specific pep¬tide phage. Such problems can be avoided by reducing the number of selection cycles and the identification of positive phage by direct immunoscreening of a large number of selected clones transferred to nitrocellulose membranes. More than one patient serum can be used for the immuno¬screening step to identify most of phage displaying epitopes recognized by disease-specific antibodies.
4.3.3. Counter-screening with negative sera
Positive clones are finally screened using different sera from normal individuals. Phage clones that are not recog¬nized by most of the sera not related to the specific disease but react with antibodies in patient?s sera can then be iden¬tified and characterized.
Using the above strategy, Minenkova et al. [99] screened several phage libraries displaying random peptides fused to the N-terminus of the major coat protein g8p against nega¬tive and positive sera for HCV [99]. Several peptides that bind HCV-specific antibodies were identified [99]. Syn¬thetic peptides with similar sequences to those displayed on the phage lost their ability to detect HCV antibodies. To overcome this difficulty, the selected peptides were ex¬pressed as octabranching multiple antigen peptides that sim¬ulated display of multiple copies of the peptide on the phage surface [100 ?102]. The multimeric synthetic peptides re¬tained their specificity and were used to develop a diagnos¬tic assay to detect HCV antibodies in serum [99].

Similarly, Kouzmitcheva et al. [103] used antibodies from a panel of human sera from patients with Lyme disease to affinity select peptide epitopes from 12 large random phage-peptide libraries. They identified 17 peptides with a diagnostically useful binding pattern: reactivity with at least three positive sera and no reactivity with any of the negative sera. Again, the method used to discover these peptides did not require any knowledge of the pathogen and involved generic procedures that are applicable to any emerging infectious disease for which no pathogen has yet been iden¬tified.
5. Selected therapeutic applications of phage display
Phage display has allowed the isolation of numerous proteins and peptides of therapeutic significance. Phage display provides access to a vast untapped pool of human monoclonal antibodies with antitumor and antiviral activi¬ties. When combined with toxins or radio-isotopes, scFv antibodies can be advantageous for cancer immunotherapy because their small size allows greater tumor penetration and faster clearance rates [67]. Fabs isolated against viral pathogens provide research tools, diagnostics and potential pharmaceutical reagents for the prophylaxis and treatment of viral infections [104,105]. On the other hand, random phage peptides selected against antibodies from HIV-in¬fected individuals uncovered immunogenic epitopes that behave as antigenic mimics of gp120 and gp41 HIV pro¬teins and, therefore, have the potential for use as broadly protective HIV-1 vaccine candidates [106]. Peptides se¬lected by phage display have also been used to bind inte¬grins, cell surface receptors necessary for anchorage and regulation of cell differentiation and migration, to prevent tumor invasion [107] and platelet aggregation [108].
5.1. Recombinant ScFv antibodies with antitumor activities
Human antibodies can be obtained by combining immu¬nization of transgenic mice containing human immunoglob¬ulin genes and classic hybridoma technology [109,110]. However, selection of human recombinant antibodies from antibody libraries displayed on phage eliminates the time required for immunization and permits methods aimed at optimizing the affinity of the selected antibodies. Huls et al.
[111] isolated an scFv antibody fragment that recognizes a tumor-associated antigen Ep-CAM from a semisynthetic (nonimmune) phage antibody library. Selection was per¬formed by subtractive selection of scFv antibody fragments on intact, labeled colorectal tumor cells in the presence of an excess of absorber cell line. Flow cytometry was then used to isolate labeled cells and cell-bound phages were eluted and propagated. To increase the half-life of scFv fragment in circulation, the isolated scFv fragment was converted into an intact human IgG1 by insertion into mammalian expres¬sion vectors containing the genes encoding human immu¬noglobulin constant regions [4]. The purified IgG1 had a low nM affinity and mediated tumor cell death [111].
5.2. Inhibition of prion propagation using Fab antibodies
Prions are the infectious agents believed to cause neuro¬degenerative diseases such as scrapie and bovine spongi¬form encephalopathy. Direct interaction between the infec¬tious and the endogenous forms of prion protein is suggested to drive the formation of the infectious isoform [112]. Therefore an antibody that can bind either form of the protein is suggested to inhibit the generation of the infec¬tious prion conformer [113]. A Fab antibody that recognizes adefined region on the cellular form of prion was selected from phage display libraries prepared from prion-deficient mice immunized with infectious prions [114]. Treating prion-infected neuroblastoma cell cultures with the isolated antiprion Fab abolished conversion of endogenous prion to the infectious protein [113]. These Fab fragments could be converted into whole IgG molecules and may play a role in treating prion diseases [113].
6. Recent innovations in display technology
6.1. Selectively infective phage (SIP)
In phage display, all phage particles remain infective as long as they have the wild type g3p (pIII) expressed at the tip of the phage. Therefore, the DNA of both specifically and nonspecifically bound phages enter bacterial cells by infection after elution from the ligand. Major tasks for phage display are to minimize adsorption of nonspecific phage to the ligand [115] and to enrich phage displaying high-affinity molecules over abundant low affinity binders [48]. In SIP technology [116], the N-terminal domains of g3p are replaced with the gene for a peptide or a protein leading to the generation of noninfective phage particles [117,118]. The missing N-terminal domains necessary for infection are supplied within adapter molecules consisting of the ligand (protein, peptide, or a small organic molecule) covalently coupled to these N-terminal domains. When the noninfective phage and the adapter molecules are mixed, infectivity is restored only to phage particles displaying protein or peptide that are capable of binding the ligand with the latter providing the missing N-terminal domains of g3p to the phage. SIP technology is a low background procedure that eliminates the need for the inefficient physical separa¬tion of specific binders from the nonspecific ones and is therefore suggested as an efficient and rapid procedure to select for high affinity interactions [116].

H.M.E. Azzazy, W.E. Highsmith Jr. / Clinical Biochemistry 35 (2002) 425? 445 439
Table 4
List of BsAbs with Potential Diagnostic Significance
Method used for generating the BsAb First Antigen (Reporter Enzyme) Second Antigen (Target) Reference
Hybridoma-hybridoma fusion Cross-linking of two Mabs to form bispecific tetrameric antibody complex Fusing hybridoma to splenocytes Hybridoma-hybridoma fusion Hybridoma-hybridoma fusion Bispecific F(ab�)2 generated by disulfide bond exchange between IgG1 Mabs ScFv diabody ScFv diabody ScFv diabody Urease Alkaline phosphatase Horseradish peroxidase (HRP) HRP �-galactosidase Alkaline phosphatase �-galactosidase �-galactosidase �-galactosidase HCG Native human erythropoietin Human lymphotoxin Biotin Follitropin Human TSH Hen-egg lysozyme Carcinoembryonic antige

I redditi di lavoro autonomo sono compensabili con le perdite di un impresa individuaele in contabilità semplificata?

Lo cito nella descrizione: escluso art.26 c. 3?

Ho verificato ed è quello...

Grazie mille

Devo stornare una fattura emessa nel 2004 per errata fatturazione...

E' sufficiente fare una nota di credito comprensiva di iva o ci sono complicanze?

Grazie

Il praticante dottore commercialista, terminata la pratica e finchè non ha passato l'esame può essere ancora inquadrato ( il rapporto che lega il praticante al dominus) come rapporto di formazione professionale e perciò non soggetto al versamento dei contributi INPS???

QUalcuno conosce riferimenti normativi e/o dottrinali???

Grazie....

1 E' fattibile ed affidabile dare lavoro ai procacciatori di affari ?
Meglio che ti affidi a contratti di lavoro occasionale o contratti co co pro
2 Questi procacciatori devono avere una partita iva loro oppure a lavoro terminato mi possono fare una ritenuta d'acconto ?
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3 E' vero che posso operare con loro (se sprovvisti di partita iva) per non più di 30gg e non possono ricevere un compenso superiore a 5000 ? ?
Contratto di lavoro occasionale, si è così senza oblbigo inps
4 Nel caso sarebbe fattibile la cosa, devo fargli una lettera d'incarico oppure basta dargli i campionari ed una volta che ricevono ordini dai vari negozi gli do solo la loro percentuale e basta ?
Vai dal commercialista e fatti fare un contratto standard e poi personalizzeria con i vari collaboratori
5 Se per esempio vanno in un negozio che vuole fargli un'ordine, loro non possono firmare col loro nome la copia commissione vero ? E se così fosse io cosa posso fare per ricevere l'ordine del negozio ???
Boh....

In che senso?