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1068 NATURE BIOTECHNOLOGY VOLUME 16 NOVEMBER 1998

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The screening of phage-displayed random peptide libraries on puri-fied receptors is a straightforward procedure for identifying pep-tides that bind to a wide variety of target molecules (e.g., monoclon-al antibodies or hormone receptors)1. More laborious selectionstrategies are required when the target receptor is present within acomplex mixture, such as whole serum2–4, cell-surfaces5, or animalorgans6. Panning a phage library on such complex receptor systemsselects a large pool of peptides that bind to the myriad differentavailable receptor molecules. Successive selection, screening, andcounterscreening steps are then required to identify the ligands forthe receptor of interest, which usually constitute a small minority ofthe peptides present in the pool. One example of this type of systemis the selection of peptides binding to disease-specific antibodies(DS-Ab). Simultaneous selections of the same phage library usingserum samples from various patients and normal individuals gener-ate pools of phage that bind to the antibodies present in each serum.Ligands for DS-Ab (DS-peptides) can be identified by isolatingthose peptides that bind specifically to immunoglobulins present ina variety of patients’ sera relative to control sera. In order to simpli-fy this laborious procedure, we developed a DNA-based selectionstrategy. Each phage-displayed peptide in a library is physicallyassociated with its encoding gene, and in this experimental systemeach peptide sequence is encoded by a unique nucleotide sequence.This unique correspondence can be used to identify peptides thatare common or different among given sets of phage by positively ornegatively selecting for their peptide-coding sequences. This goalcan be quickly and efficiently achieved by exploiting the specificityand affinity of nucleic acid hybridization reactions and applying tophage display some of the high-throughput and sensitive methodsdeveloped in the field of DNA analysis and genomics. We have usedthis DNA-based screening procedure to select ligands for antibodiesthat are specifically associated with hepatitis C virus7 (HCV) infec-tion in humans, a major cause of chronic liver disease worldwide.

ResultsDNA-based selection of HCV-specific peptides. A random peptidelibrary (RPL) displayed on phage was panned on the C12 and C16

sera from HCV-infected patients, generating phage pools indicatedas P12 and P16, respectively. To identify the peptide-codingsequences common to both these pools, single-stranded DNA(ssDNA) was derived from P12 pool, whereas a population ofbiotinylated oligonucleotides of negative polarity containing thepeptide-coding sequence was derived from P16 pool (Fig. 1A). P12ssDNA was hybridized to the biotinylated P16 oligonucleotides andthe hybridized complexes were captured on streptavidin. Thehybridized P12 ssDNA was then recovered and used to transformbacterial cells, thus obtaining the phage pool designated asP12·P16. Phage pools P12, P16, and P12·P16 were assayed byELISA for reactivity with sera C12 and C16 (Fig. 1B). Relative tothe reactivity of serum C16 with the P16 phage pool, the P12 phageshowed 25% reactivity with the same serum. In contrast, the reac-tivity of the P12·P16 phage with the same C16 serum increased toabout 85%, whereas the reactivities of the P12 and P12·P16 poolswith serum C12 were not significantly changed.

A further selection step was then carried out by hybridizing theamplified P12·P16 pool with the P17 pool obtained by affinityselecting the RPL with C17 serum from an HCV-infected patient.Relative to the reactivity of the serum C17 with the P17 pool ofphage, the P12·P16 pool showed a 25% reactivity. In contrast, thereactivity of the (P12·P16)·P17 pool with the same serum increasedto about 85% of the P17 reactivity.

DNA-based screening of individual clones. Binding of serumantibodies to a phage-displayed peptide can be conveniently substi-tuted by the hybridization between a probe derived from the pool ofpeptide-coding sequences enriched by the serum and the peptide-coding sequence of the individual clone. Peptide-coding sequencesfrom 88 clones derived from the (P12·P16)·P17 pool were individu-ally amplified by PCR and gridded in replica onto nylon mem-branes (screening S19). The immobilized DNA on each membranewas then hybridized to a series of radioactively labeled DNA probes;each probe was derived from the peptide-coding sequences of thephage pool P12, P16, P17, P14, or P65 (which were in turn obtainedby panning the RPL on the positive sera C12, C16, C17, C14, andC65, respectively). A positive signal on the filter identifies clones

DNA-based selection and screening ofpeptide ligands

Fabrizia Bartoli, Maurizio Nuzzo, Lorena Urbanelli, Francesca Bellintani1, Caterina Prezzi, Riccardo Cortese, and Paolo Monaci*

Istituto di Ricerche di Biologia Molecolare P. Angeletti Via Pontina km 30.600 00040 Pomezia (Roma), Italy. 1Kenton, I.R.C.C.S. S. Lucia Via Ardeatina 306 -00173 Roma, Italy. *Corresponding author (e-mail: [email protected]).

Received 23 February 1998; accepted 3 September 1998

Phage display selection strategies rely on the physical link between the displayed heterologous pro-tein ligand and the DNA encoding it. Thus, genes expressing a ligand with a specific binding affinity canbe selected rapidly. To improve the specificity and sensitivity of this technology for potential use in iden-tifying ligands to a specific antibody present in a complex mixture, we incorporated a DNA selection stepalong with the phage display technology. Ligands for hepatitis C virus (HCV) antibodies present in serumwere identified by panning a phage-displayed random peptide library against pools of serum HCV anti-bodies. An additional DNA hybridization screening step using single-stranded DNA isolated from one ofthe pools increased the specificity and sensitivity, resulting in the selection of an HCV antibody ligandwith diagnostic potential.

Keywords: phage display, HCV, diagnostics, PCR

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that are present in the phage pool from which the probe wasderived. All of the 88 clones examined scored positive with at leasttwo different probes, whereas they did not hybridize with either ofthe two probes derived from selection of the RPL with a mixture of10 negative sera (from uninfected individuals, data not shown).DNA sequence analysis of the 88 clones identified 13 different pep-tide-coding sequences. Culture supernatants from each of these 13clones were prepared and their ELISA reactivity was tested with 16positive and 16 negative sera (clones P19XX; Fig. 2). The frequencyof reactivity with positive sera was high for all clones, ranging from19–100%, whereas no reaction was obtained with any of the nega-tive sera tested (data not shown). Each of the clones displayed a sta-tistically significant selectivity for positive sera and were thus classi-fied as HCV-specific phage (HCV-phage).

DNA-based depletion of clones identified by the first screen-ing. To identify new ligands we depleted the 13 clones identified bythe first screening from the pool using a DNA-based protocol. An

ssDNA pool was derived from the (P12·P16)·P17 pool,and a pool of biotinylated oligonucleotides was gener-ated from the pool of 88 screened clones (indicated asPs19). After hybridization and streptavidin capture ofhybridized complexes, the nonhybridized populationwas recovered. Following amplification, the depletedpopulation was subjected to a second round of deple-tion and the resulting pool of clones was analyzed byDNA screening (screening S32). Hybridization usingthe Ps19 probe scored only four positives out of 88clones, indicating a depletion efficiency higher than95%. As expected, DNA-screening experiments usingthe probes derived from pools P12, P16, P17, P14, andP65 revealed a lower frequency of reactivity, whereasnone of the clones examined was scored by a probederived from selection of the RPL with a mixture of 10negative sera (data not shown). Forty-eight clones dis-playing reactivity with at least two probes were ana-lyzed by sequencing and found to represent 19 differ-ent sequences none of which were represented amongthe 13 clones obtained from selection S19. Culturesupernatants from each of these 19 clones were pre-pared and their ELISA reactivity tested with 16 differ-ent positive and 16 negative sera. Only 10 clones dis-played statistically significant reactivity with positive

sera and were therefore considered as HCV-specific (clonesP32XX; Fig. 2).

DNA-based detection of anti-HCV antibodies. A mixture ofeight HCV-phage was incubated with positive and negative sera ina PCR plate that was coated with protein A. After incubation, theunbound phage were washed away, and phage that had been cap-tured by serum antibodies were detected through PCR amplifica-tion of the region of the phage genome coding for the displayedpeptide. Amplification was preceded by a short incubation at 95°Cthat disrupted the phage capsid, thus providing a built-in “hotstart” to the PCR8. Reaction products were analyzed by agarose gelelectrophoresis. A DNA fragment of the expected size was detectedin all reactions in which positive sera were assayed, whereas no PCRproducts were observed when negative sera were used (Fig. 3A).

To detect the individual reactivity of the phage present in theHCV-phage mixture, synthetic oligonucleotides bearing the pep-tide-coding sequences of selected clones were immobilized on a

dsDNA fromP16 pool

b

ssDNA fromP12 pool

hybridization

b

elution andtransformation

P12·P16 ssDNA

b

streptavidin capture ofhybridized complexes

(+) (-)

bead

Figure 1. (A) DNA-based selection scheme. (B) ELISA reactivity of phage pools derived from DNA-based selection with positive sera C12 (whitecolumn), C16 (hatched column) and C17 (black column).

A

0.5 < A ≤ 1.0 1.0 < A ≤ 1.5 A ≤ 0.3 0.3 < A ≤ 0.5 A >1.5

mapping clone C12 C16 C17 C14 C65 C13 C15 C18 C19 C22 C39 C40 C45 C47 C56 C57 p

core-1 P1905 < 0.001

P1909

P1913

P1972

P1976

P3206

P3218

core-2 P1901c

core-3 P1902 0.032

P1904

P1914c

P1977

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P3234

NS4b P1929

P1967

P1981c

P3202

P3213

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P3243

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0.006

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0.068

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0.006

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Figure 2. ELISA reactivity of HCV-phage with positive sera.

0.0

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P12 P16 P17 P12·P16 (P12·P16)·P17phage pool

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nylon membrane. The phage-PCR product derived from theamplification reaction with positive serum was radioactivelylabeled and used to probe the nylon membrane. This experimentdetected antibodies binding to phage P1909, P1901c, P1977,P1904, P1981c, P1929, and P3243 in serum C17 (Fig. 3B).Similarly, antibodies binding the phage-borne peptides P1909,P1904, P1914c, and P1981c were detected in serum C22 (Fig. 3C).These data match the results of the ELISA in which single cloneswere tested. In this case, however, the ratio between the positive sig-nals and the cutoff value was much larger than that of the ELISA;this also allowed the detection of antibody binding to clone P1901cthat could not be scored by ELISA.

Mapping of HCV-phage. The phage-borne peptides were usedto affinity-purify antibodies from a positive serum. These antibod-ies were then tested in ELISA on the other phage to identify clonesmimicking the same determinant, as well as on recombinant HCVproteins, to map the mimicked viral epitope. Antibodies affinitypurified from a positive serum using clone P1905, P1909, P1913,P1972, P1976, P3206, or P3218, specifically cross-reacted withP1905 and P1909, indicating that all seven phage-borne peptidesdetect antibodies with the same specificity (Fig. 4A). In contrast,no reactivity was found against either wild-type (wt) clone or otherHCV-phage, confirming that the other HCV-phage detect antibod-ies with different binding specificity. No reactivity was detectedwhen wt phage were used, or when antibodies were affinity puri-fied from a negative serum (data not shown). Antibodies purifiedby clone P1905 specifically recognized the HCV-core fragmentspanning amino acids (aa) 1–72, expressed in bacteria as a fusionwith glutathione S-transferase (GST-HCV 1-72; Fig. 4B). The sameexperiments in which clones P1909, P1913, P1972, P1976, P3206,or P3218 were used as immunoadsorbents gave similar results(data not shown), indicating that all seven phage detect antibodies

that bind the N-terminus of the core protein. Alignment of theirpeptide sequences reveals a consensus, which displays a similaritywith the region of the natural antigen from aa 10–18, designated ascore-1 region (Fig. 4C).

Through a similar experimental procedure most of HCV-cloneswere mapped to four distinct viral regions. Similarities between theconsensus sequence of each group and the corresponding viralregion were revealed (Fig. 4C).

Earlier diagnosis of HCV infection. We tested the reactivity ofclone P1909 on a group of serial bleeds from an individual plasmadonor during seroconversion and compared it with the resultsobtained from a licensed HCV screening test. Clone P1909 revealedseroconversion starting from the third bleed 2 weeks before the ref-erence ELISA test (Table 1).

Assessment of reactivity of otherwise indeterminate serumsamples. By using presently available diagnostic assays for anti-HCV antibodies, sera C86, C88, and C94 displayed a unique reac-tivity with core antigen (c22p; 2+, 3+, and 4+, respectively). Thesesamples were therefore considered indeterminate, as their reactivi-ty may reflect nonspecific IgG binding. The same sera were testedagainst HCV-phage that mimic distinct core regions: for eachserum, multiple reactivities against distinct core epitopes weredetected, which identifies these sera as being positive (Table 2).

Detailed dissection of humoral response. Clones binding anti-bodies directed against the same short viral region displayed over-lapping, but quite different ELISA reactivity profiles. For example,clone P1904 and P1977 mimicked the same core-3 region; however,they displayed some specific reactivities with the positive sera test-ed. Phage P1904 reacted with serum C14 but not with serum C18,and the reverse was true for phage P1977 (Fig. 2). These reagentscan therefore discriminate among classes of antibodies that recog-nize the same antigen sequence.

Figure 3. Detection of HCV-specific serum antibodies by phage-PCR immunoassay. (A) Lane M: DNA molecular size marker; lanes 1–12:amplification products derived from assays using 11 positive; and lanes 13–22: 10 negative sera. The arrow indicates the size of the expectedamplification product. Products of a phage-PCR immunoassay using positive serum (B) C17 and (C) C22 were analyzed by hybridization to theindicated peptide-coding sequences. Hybridization signals are indicated by black bars (scale on bottom abscissa). ELISA reactivities of listedphage with HCV sera are indicated by white bars (scale on top abscissa).

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DiscussionSera from patients affected by a given disease often contain a largenumber of DS-Ab: their detection and characterization normallyrequires the natural antigen, a serious limitation when this is notreadily available or is simply unknown, as is the case for manyautoimmune diseases. We demonstrate that a DNA-based selection

strategy that exploits the link between the phage-displayed ligandand its DNA coding sequence can be used to identify peptides thatdetect DS-Ab. These specific ligands can be selected using only serafrom clinically characterized patients, without the availability of, orinformation regarding, the natural antigen.

This method requires only very small quantities of serum,allowing the study of clinically characterized patients from which alimited amount of serum is usually available. It also permits an effi-cient subtractive procedure, which is in contrast to other strategiesfor identifying DS-peptides that were confined to positive selec-tion2–4. DNA-based selection relies on the much higher affinity ofDNA–DNA interaction than that between serum antibodies andphage-borne peptides. In addition, the molar ratio between thebiotinylated oligonucleotide and ssDNA can be much higher thanthat obtained between phage and serum antibodies, and this sub-tractive process can be repeated several times and performed inmany different ways. DNA hybridization also allows large numbersof clones to be screened rapidly, with a sensitivity higher than thatof immunological techniques.

The DNA label does not interfere with the interaction betweenthe phage-displayed ligand and the target molecule, as the DNAsequences are located within the phage envelope during ligand–tar-get interaction and can be amplified only upon heat-mediated dis-ruption of the viral capsid, also ensuring the specificity of theamplification. The sensitivity and versatility of this phage-PCRassay is greater than the conventional enzyme-based immunoas-says, and can be further extended using alternative detection meth-ods for PCR products9,10.

We used this procedure to select several ligands for antibodiesspecifically associated with HCV infection. Most of the selectedpeptides specifically bind serum antibodies directed against HCVB-cell epitopes located in the core and NS4b regions, which areknown to contain immunodominant epitopes11,12. We did not selectpeptides mapping to the immunodominant HCV-NS3 protein,

Table 1. Test of an HCV-seroconversion panel.

Sample Days since P1909 Referenceno. first bleed ELISA HCV ELISA

1 0 0.2 0.52 5 1.1 0.63 7 2.5 0.54 12 3.2 0.75 14 2.8 0.86 19 3.3 1.67 21 4.0 2.18 26 4.6 2.5

ELISA data are expressed as the ratio between the sample absorbance and thecutoff value.

Table 2. Discrimination of indeterminate samples by HCV-coremimics.

Human seraMapping Clone C86 C88 C94

core-1 P1909 ++++ +++ ++++core-2 P1901c ++ - +++core-3 P1904 ++++ - ++++core-3 P1914c ++ - -core-3 P1977 - ++ ++++

-: A≤0.3, +: 0.3<A≤0.5, ++: 0.5<A≤1.0, +++: 1.0<A≤1.5, ++++: A>1.5

Figure 4. Characterization of HCV-phage. (A) ELISA reactivity of antibodies affinity-purified from a positive serum (C65 for P3218 and C17 for theother clones) using the phage indicated in abscissa, tested against wt phage (white column), P1901c (dark grey column), P1904 (checkedcolumn), P1905 (black column), P1909 (hatched column), and P981c (pale grey column). (B) ELISA reactivity of antibodies affinity-purified usingthe phage indicated in abscissa from a positive serum (C65 for P3218 and C17 for the other clones) against GST (white column), GST-HCV 1-72(black column), GST-HCV 1186-1647 (pale grey column), GST-HCV 1649-1904 (hatched column), GST-HCV 1679-1711 (dark grey column) andGST-HCV 1904-1959 (checked column) proteins. (C) Sequences of HCV-peptides and partial sequences of the HCV core and NS4b proteins(subtype 1b [ref. 19]) are reported. Homologies of each clone with the viral sequence are highlighted by grey boxes, whereas similarities of eachclone to the consensus sequence of the group are indicated in bold.

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known to contain discontinuous epitopes12, suggesting that mimicsof linear epitopes predominated in library screening.

The selected ligands have potential for use in diagnostic assays.The HCV-phage detect anti-HCV antibodies in patients’ serumwith a sensitivity comparable to, or even higher than, that of thecorresponding region of the natural antigen. This sensitivity is theresult of selecting those peptide structures that best bind HCV-spe-cific antibodies, and of the multivalent peptide display on thephage. The use of highly specific ligands outside the context of thenatural antigen reduces the incidence of unspecific reactivities.Detecting specific antibodies by using mimics of different epitopesfrom the same antigen greatly enhances the resolution power ofthis analysis, by reducing the frequency of indeterminate samplesand allowing a detailed dissection of the polyclonal responseagainst the same epitope. The correlation of such typing to thegenetic background or the clinical history of the patient is expectedto improve our understanding of the immune response to viralinfection.

Experimental protocolAffinity selection. Construction of phage-displayed RPLs has beendescribed13,14. The human sera used in this study were collected and character-ized as described3. Positive sera were used to screen 2×1011 transducing unitsof phage that comprised an equimolar mixture of the pVIII9aa and pVIII9aa-cys libraries. For each serum, two rounds of affinity selection were performedas previously described3. Similarly, a mixture of 10 different negative sera wasused to affinity select the same library mixture. Anti-HCV seroconversionpanel was obtained from Boston Biomedica (PHV903; Bridgewater, MA) andtested by the second-generation HCV enzyme immunoassay test (AbbottLaboratories, Chicago, IL).

ELISA using culture supernatants and human sera. Multiwell plates(Immunoplate Maxisorp; Nunc, Roskilde, Denmark) were coated overnightat 4°C with the anti-pIII monoclonal antibody 57D1 (ref. 15) at a concentra-tion of 1 µg/ml in 50 mM NaHCO3, pH 9.6. After washing several times withphosphate buffered saline (PBS) containing 0.05% Tween-20 (PBS/Tween),plates were incubated for 60 min at 37°C with ELISA blocking buffer (5%nonfat dry milk in PBS/Tween). A 1:1 dilution of culture supernatant con-taining phage (prepared from XL1-blue infected cells as described in ref. 14)in ELISA blocking buffer was added to each well and allowed to bind for 1 hat 37°C. Human serum was incubated for 30 min at room temperature with 5×1011 plaque forming units/ml of phage f11.1 (ref. 15), 20 µl/ml of XL1-bluecell protein extract3 and 10 µl of supernatant from unrelated rat hybridomacells3 in ELISA blocking buffer. A 1:400 serum dilution was used when phagepools were tested, a 1:100 dilution was used when single clones were tested,and a 1:20 dilution when clone P1909 was tested with anti-HCV seroconver-sion panel. Plates were rinsed with PBS/Tween, and 200 µl of the preincubat-ed serum mixture were added to each well; incubation continued for 2 h atroom temperature (phage pools and seroconversion panel), or overnight at4°C (single clones). Plates were then washed with PBS/Tween, and 200 µl/wellof goat antihuman IgG (Fc specific) alkaline-phosphatase conjugated anti-bodies (Sigma, St. Louis, MO) diluted 1:5000 in ELISA blocking buffer wereadded to each well. After a 60-min incubation at room temperature, plateswere washed and alkaline phosphatase activity was detected by incubationwith 1 mg/ml solution of p-nitrophenyl phosphate in ELISA substrate buffer(10% diethanolamine buffer, 0.5 mM MgCl2, pH 9.8). The plates were read byan automated ELISA reader (Labsystems Multiskan Bichromatic, Helsinki,Finland) and the results were expressed as A=A405nm – A620nm. ELISA datareported in display items are average values from two independent assays.Error bars indicate the standard deviation from the mean of each value.Values reported in Figure 2 were considered statistically significant if theywere greater than the 0.3 cutoff and differed more than 3s (s={1/2[s2p+s2w]}1/2) from the background signal observed for the wt phage. The pvalue in the same figure is the probability that the observed frequency distrib-utions of reactivities of the positive and negative sera are statistically the sameaccording to the x2 test. Cutoff value in Table 1 was calculated as the meanvalue of negative control samples plus 0.6 absorbance units.

DNA-based selection. XL1-blue bacterial cells were infected with the givenphage population and plated onto L-agar (1% Bacto-tryptone, 0.5% yeastextract, 1.5% Bacto-agar, 0.170 M NaCl, pH 7.2) plates containing 100 µg/mlampicillin, 1% glucose. After overnight incubation at 37°C, culture super-

natants were prepared from infected, ampicillin-resistant colonies asdescribed13. Phagemid ssDNA was purified as described16. XL1-blue bacterialcells were infected with the given phage population and plated onto L-agar,100 µg/ml ampicillin, 1% glucose plates. After overnight incubation at 37°C,bacterial colonies were scraped, resuspended in L-broth (1% Bacto tryptone,0.5% yeast extract, 0.085 M NaCl, pH 7.2) and double-stranded (ds) plasmidDNA was purified on Qiagen (Hilden, Germany) columns according to themanufacturer’s protocol. dsDNA was first digested with EcoRI, and therecessed 3´-termini were filled-in with Klenow polymerase using biotin-dUTP as described16 (Boehringer Mannheim, Mannheim, Germany). Thepeptide-coding sequence was then excised by digestion with BamHI, separat-ed by 15% PAGE and recovered by electroelution of the appropriate gel frag-ment16. About 50 ng (6 pmol) of this biotinylated ds fragment were incubatedwith 0.3 mg of streptavidin-coated magnetic microbeads (Dynal, Oslo) atroom temperature for 60 min. After extensive washing with 10 mM Tris-HCl,pH 7.5, 1 mM EDTA, 1 M NaCl (TE/NaCl), the beads were incubated with100 µl of 0.15 M NaOH for 15 min at room temperature. The supernatantwas discarded and the incubation repeated. The beads were washed withTE/NaCl and then with water. The biotinylated negative-strand was dissoci-ated from the streptavidin-coated beads by heating the complex for 20 min at65°C in a solution containing 10 mM Na2 EDTA, pH 8.2, and 95% HCONH2.The beads were removed, the supernatant dried, and the recovered biotiny-lated product resuspended in water. The biotinylated probe was quantified bya dot-blot assay, using a biotinylated ssDNA of known concentration as refer-ence. About 0.125 ng of the biotinylated probe were hybridized with about 2µg of the ssDNA in 20 µl of hybridization buffer (3.0 M tetramethylammoni-um chloride, 0.05 M Na2HPO4, 0.5% SDS, 1 mM EDTA) containing 5 µg ofcarrier ssDNA (derived from phagemid pBS8+ [ref. 17]) overnight at 80°C.Following incubation, the hybridization mixture was diluted in 1 ml ofhybridization buffer that had been prewarmed to 80°C. Thirty microliters ofstreptavidin-coated magnetic microbeads were added, and the mixture wasincubated for 90 min at 37°C. Nonhybridized ssDNA was removed and thebeads washed twice with hybridization buffer at 80°C. Finally, the hybridizedssDNA was eluted from the beads by addition of 0.15 M NaOH. This opera-tion was repeated, then the collected supernatant was neutralized and used totransform competent bacterial cells by electroporation16.

DNA-based screening. Peptide-coding sequences from the dsDNA derivedfrom the phage pool were amplified by PCR. The amplification reaction wascarried out in a final volume of 100 µl containing 50 ng of selected-pooldsDNA, 0.8 µM M13 (5´-GTTTTCCCAGTCACGAC-3´) and pC89 (5´-AGA-GATTACGCCAAGCC-3´) primers, 200 µM dNTPs, 7.5% DMSO, 1× Taqpolymerase buffer (50 mM KCl, 10 mM Tris-HCl, pH 9.0, 0.1% Triton X100,1.5 mM MgCl2), 1.25 units Taq polymerase (Boehringer Mannheim).Amplification was performed in 30 cycles using the following conditions: 10 sat 94°C, 10 s at 45°C, 10 s at 72°C. PCR products were digested with EcoRIand BamHI, and the digested fragments were purified by 20% PAGE andelectroelution from the appropriate gel fragment. About 3 pmol of this prod-uct were radiolabeled with a[32P]dATP and a[32P]dCTP by fill-in reactionsusing Klenow-fragment and then purified by G-50 Sephadex gel filtration16.XL1-blue bacteria were grown at 37°C in Terrific broth (1.2% Bacto-tryp-tone, 2.15% yeast extract, 0.4% glycerol, 0.01 M KH2PO4, 0.07 M K2HPO4)containing 20 µg/ml tetracycline to an A600nm =0.150–0.250 (as measured at a1:10 dilution). The cells were then infected with the amplified phage from aDNA-selection and plated on L-agar containing 100 µg/ml ampicillin, 1%glucose. After overnight incubation at 37°C, 88 bacterial colonies were usedto inoculate wells of an ultraviolet-sterilized ELISA multiwell plate contain-ing 200 µl of L-broth added with 100 µg/ml ampicillin and 1% glucose. The88 colonies from the master plate were used to inoculate a PCR multiwellplate (Thermowell; Corning Costar, Cambridge, UK) using a 96-pin griddingdevice (‘Q’ rep, Genetix, Dorset, UK). The peptide-coding sequence of eachclone was amplified by PCR using a reaction mixture containing: 0.8 µM F24(5´-TTGCAGGGAGTCAAAGGCCGCTTT-3´) and E24 (5´-GCTACC-CTCGTTCCGATGCTGTCT-3´) primers, 250 µM dNTPs, 7.5 % DMSO, 1×Taq polymerase buffer, 1.25 units Taq polymerase in total volume of 80 µl.Amplification was programed for 30 cycles (10 s at 94°C, 10 s at 60°C, and 10s at 72°C). The synthetic oligonucleotides used in this study were obtainedfrom Genset (Paris). About 1 µl of the PCR amplification product (diluted in5× SSC) was spotted onto a nylon membrane (Hybond N+; Amersham,Buckinghamshire, UK) under vacuum by using a Dot-Blot apparatus (Bio-Rad, Hercules, CA). The membrane was incubated with high-salt denaturingsolution (0.5 M NaOH, 1.5 M NaCl) for 7 min and then with neutralizingsolution (0.5 M Tris-HCl, pH 7.4, 1.5 M NaCl) for 3 min. DNA samples were


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then fixed to the membrane by placing it on filter paper that had been soakedin 0.4 M NaOH, twice for 20 min. The membrane was then rinsed by immer-sion in 5× SSC, and finally prehybridized in 5× SSC, 5× Denhardt’s solution,plus 0.1% SDS and 100 µg/ml salmon sperm DNA at 65°C for at least 4 h. Thedenatured probe was added to the DNA-hybridization solution comprising5x Denhardt’s solution, 5× SSC, and 0.1% SDS, and incubated overnight at65°C. Following hybridization, the membrane was washed with 0.1× SSC and0.1% SDS at 72°C. Hybridization signals were quantified by aPhosphorImager (Molecular Dynamics, Sunnyvale, CA). Sequences of thepositive clones were determined as described18.

DNA-based depletion. About 5 pmol of the biotinylated ssDNA derivedfrom the mixture of the 88 clones identified by S19 screening were hybridizedovernight at 80°C with about 2 fmol of the ssDNA population P12·P16·P17 in20 µl of hybridization buffer (3.0 M TMACl, 0.05 M Na2HPO4, 0.5% SDS, 1mM EDTA), containing 0.01 pmol ssDNA carrier. The hybridized complexeswere diluted in 1 ml of 80°C prewarmed hybridization buffer, and capturedby adding 50 µl of streptavidin-coated magnetic microbeads (90 min at37°C). Nonhybridized ssDNA was recovered. Additional washes at 80°C withhybridization buffer were carried out, and recovered material was pooled.Recovered ssDNA was concentrated by isopropanol precipitation and resus-pended in water. The recovered ssDNA was amplified by inverse PCR in areaction mixture containing: 1 µM Amp-3´out (5´-GGTTAGCTCCTTCG-GTCCTCCGATC-3´) and Amp-5´out (5´-GCTTTTTTGCACAA-CATGGGGGATC-3´) 5´-phosphorylated primers, 200 µM dNTPs, 1.25 unitscloned Pfu polymerase (Stratagene, La Jolla, CA), 1x Pfu polymerase buffer,20% glycerol, in a total volume of 100 µl. Amplification was programed for 30cycles (30 s at 99°C, 20 min at 68°C). PCR-amplification product was EtOH-precipitated and resuspended in 20 µl of a ligase reaction mixture containing:2× T4 DNA-ligase buffer and 5 units of T4 DNA-ligase (BoehringerMannheim). Ligation was performed for 30 min at room temperature. Theligation product was used to transform competent bacterial cells by electro-poration, and culture supernatants containing phage were prepared.

Phage-PCR immunoassay. A polycarbonate, multiwell PCR-plate(Thermowell) was coated overnight at 4°C with 50 µl/well of Protein A(Pharmacia, Uppsala, Sweden) at a concentration of 0.5 µg/ml in 50 mMNaHCO3, pH 9.6 . After washing with PBS containing 1% Triton X100(PBS/Triton), the wells were filled with 3% bovine serum albumin inPBS/Triton (blocking buffer) and incubated for 120 min at 37°C. A mixtureof 8 HCV-phage (P1901c, P1904, P1909, P1914c, P1929, P1977, P1981c, andP3243) containing 2.5×108 particles of each clone was prepared. A 50 µl mix-ture containing 0.5 µl serum, the phage mixture and a 10-fold excess overtotal phage of carrier phage f1R1 in blocking buffer was added to each well.Phage particles were allowed to bind at 37°C for 40 min. The plate was thenwashed with PBS/Triton and phage particles were detected by PCR-amplify-ing their peptide-coding sequences. Next, a 50-µl mixture containing 0.75µM IP-for (5´-CTGTCTTTCGCTGCTGAGGGTGAATTC-3´) and IP-rev (5´-GTCACGACGTTGTAAAACGACGGCCAG-3´) primers, 200 µM dNTPs, 1xTaq polymerase buffer, and 1.25 units Taq polymerase was assembled. Inorder to disrupt the phage capsid, amplification was preceded by a 5 minincubation at 95°C. Then 22 cycles were programed (10 s at 94°C, 10 s at72°C) at the end of which a final extension step (1 min at 72°C) was carriedout. The reaction products were electrophoresed through a 2.5% agarose geland visualized by staining with ethidium bromide. About 1 pmol of thephage-PCR immunoassay product of the C17 and C22 sera reactions wasend-labeled with g[32P]dATP, using T4 polynucleotide kinase (New EnglandBiolabs, Beverly, MA). Three picomoles of synthetic oligonucleotides bearingthe peptide-coding sequences of phage P1901c (5´-GCTCGGTTTA-GAGCCAGGAAACTTCCC-3´), P1904 (5´-GATCCGAAGGTGCAC-CACGCCTTGGAAGCAGG-3´), P1909 (5´-GATCATTCGTATTACGCCGT-GTCTTG-3´), P1914c (5´-CGATAGAGAGGGACCGTGAGCTGGTAG-3´),P1929 (5´-CCAAAAGTGGGTCGGAGGGGCTGTCGC-3´), P1977 (5´-GATCCCGGCTTCTACGCGCAAGCACGTAAGAG-3´), P1981 (5´-GTGTCTTGAAGAAATATAATGGGTGGG-3´), P3243 (5´-CTGAGCGTTGATGCGGTGAGTTGGGCT-3´) were spotted in duplicateonto a 5× SSC prewetted nylon membrane. Control oligonucleotides com-prised 0.1 pmol of the F24 synthetic oligonucleotide 5´-TTGCAGGGAGT-CAAAGGCCGCTTT-3´, which encodes part of the amplified region of thevector (positive control), as well as 3 pmol of an oligonucleotide that encodes

an unrelated peptide (negative control; 5´-CGGCTTGTTGGCAGAGCG-CATGCTCTT-3´). Hybridization signals were quantified by aPhosphorImager. Data reported in Figure 3 are average values from two inde-pendent assays, with error bars indicating the standard deviation from themean of each value.

Mapping of HCV-phage. Sequences of HCV-polypeptide (subtype 1b [ref.19]) were amplified by PCR using suitable primers and cloned into the bacte-rial expression vector pGEX-3X20. The recombinant fusion proteins linked toGST were expressed in E. coli and affinity-purified as described20,21.Recombinant proteins are indicated as GST-HCV x-y, where x and y refer tothe position of the N-terminal and C-terminal amino acids of the HCV frag-ment, respectively. Amino acid positions of the HCV polypeptide are num-bered as predicted from the nucleotide sequence of the viral genome19. Thepanel of recombinant proteins tested included GST-HCV 1-72, GST-HCV1186-1647, GST-HCV 1649-1904, GST-HCV 1679-1711, and GST-HCV1904-1959. Affinity-purification of phage-specific antibodies from serumand test of their reactivity was carried out as described3.

AcknowledgmentsWe wish to thank Frank Graham and our colleagues at IRBM for reading themanuscript, making comments, suggestions, and criticisms, and J. Clench for thelinguistic revision of the text.

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5. Barry, M.A., Dower, W.J., and Johnston, S.A. 1996. Toward cell-targeting genetherapy vectors: selection of cell-binding peptides from random peptide-pre-senting phage libraries. Nat. Med. 3: 299–305.

6. Pasqualini, R. and Ruoshlahti, E. 1996. Organ targeting in vivo using phage dis-play peptide libraries. Nature 380:364–366.

7. Alter, H.J. 1995. To C or not to C: these are the questions. Blood. 85:1681. 8. Birch, D.E., Kolmodin, L., Laird, W.J., McKinney, J., Wong, J., Young, K.K.Y. et al.

1996. Simplified hot start PCR. Nature 381:445–446.9. Livak, K.J., Flood, S.J.A., Marmaro, J., Giusti, W., and Deetz, K. 1995.

Oligonucleotides with fluorescent dyes at opposite ends provide a quenchedprobe system useful for detecting PCR product and nucleic acid hybridization.PCR Methods Appl. 4:357–362.

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15. Dente, L., Cesareni, G., Micheli, G., Felici, F., Folgori, A., Luzzago, A. et al. 1994.Monoclonal antibodies that recognise filamentous phage. Useful tools for phagedisplay technology. Gene 148:7–13.

16. Sambrook, J., Fritsch, T., and Maniatis, T. 1989. Molecular cloning: a laboratorymanual 2nd ed. Cold Spring Laboratory Press, Cold Spring, NY.

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20. Smith, D.B. 1993. Purification of glutathione S-transferase fusion proteins.Methods in Molecular and Cellular Biology. 4:220–229.

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