1551 MPRRRRIRRRQK 1 Phage display (common panning) 3 PMID:21596749 3-O-sulfated heparan sulfate, 3-OS HS Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) Viral entry and glycoprotein D binding assays together with fluorescent microscopy data indicated that peptide MPRRRRIRRRQK was potent in blocking HSV-1 entry into primary cultures of human corneal fibroblasts and CHO-K1 cells transiently expressing different glycoprotein D receptors. Interestingly, peptide MPRRRRIRRRQK isolated against 3-OS HS displayed wider ability to inhibit entry of clinically relevant strains of HSV-1 and some divergent members of herpesvirus family including cytomegalovirus and human herpesvirus-8. 1552 CKHYGGGVAC CYKNVDSGGC CRFLLPQGC CYEGSEVSC CLRQGNPTC CGSGGMSPSC CSWKYWFGEC CGQWLGNWLC 8 Phage display (in vivo) 4 PMID:21683670 Mouse adipose stromal cells, ASCs Not determined. Not determined. Not determined. CX7C, CX8C, and CX9C FUSE5-based phage display library pool Comparative analysis of individual phage clones in vivo revealed peptide CSWKYWFGEC, homing to ASCs 1000 times more efficiently than to LSC, as a peptide with the highest specificity for ASCs. 1553 CAVYRSTGC CESGFPTVGC CLGVGPGFC CIRGKAGRC 4 Phage display (in vivo) 4 PMID:21683670 Mouse lung stromal cells, LSCs Not determined. Not determined. Not determined. CX7C, CX8C, and CX9C FUSE5-based phage display library pool 1554 NSSASSRGNSSSNSVY NSLRKYSKLK 2 Phage display (common panning) 4 PMID:21610657 Etoposide, VP-16 Transcription factor E2F4, E2F-4 Not determined. Not determined. T7 phage display library Biotinylated etoposide derivative was immobilized on a streptavidin-coated 96-well microplate. Etoposide (VP-16) is an anti-tumor compound that targets topoisomerase II (top II). In this study, authours had identified an alternative binding protein of etoposide by screening a library of T7 phage-displayed peptides. Peptide NSSASSRGNSSSNSVY displays similarity with the ser-rich domain in E2F-4, a transcription factor in cell cycle-regulated genes, suggesting that etoposide might interact with E2F-4 via this domain. 1555 EAHVMHKVAPRP QNTATAVSRLSP ATHTNQTHALYR VSNHKALDYPTR DSGRYSMTNHYS 5 Phage display (common panning) 3 DOI:10.1002/adma.200500863 Zinc oxide, ZnO Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) Five different sequences were determined from 35 clones. Approximately half of the selected phage viruses (18 of 35) displayed the same ZnO-1 peptide (EAHVMHKVAPRP). Comparison of the sequences of the selected peptides indicates a preponderance of amino acid residues with functional side chains. Interestingly, the ZnO-1 peptide has a greater number of basic and hydrophobic residues than the other selected peptides. The binding affinity for the monoclonal phage displaying each selected ZnO-binding peptide was estimated from the ratio of the number of bound phage viruses to the total number of phage viruses added to the ZnO-binding assay. 1556 SVSVGMKPSPRP(4)[NT] TMGFTRPRFPHY(3)[NT] SWFSYLSPWVRS(2)[NT] TWDWRMFPLRPW(1)[NT] MYLICPEQRSPL(1)[0.67] HPSVIPPSPVMS(1)[NT] HYLIGLGCIGLN(1)[NT] HKAHVSKHSVPI(1)[NT] GPLPSNHSFQHP(1)[NT] 9 Phage display (common panning) 5 PMID:10219262 CSF samples from neurocysticercosis patient #1 Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA In phage ELISA, the absorbance at 450 nm was measured. Data shown are expressed as the difference between the average A405nm values for selected phage and nonrelated phage G1. NT represents not tested. To increase the specificity of selection, plates coated with anti-human Fc antibody were used in five rounds of biopanning. Sequence similarities between affinity-selected clones and three known T. solium and T. crassiceps proteins were encountered. 1557 SHVPRIGGPNFW(2)[NT] FSVPWPTRPPHW(2)[1.28] SWNHWLYTYFPQ(2)[NT] WPYKPYWMPNFW(2)[NT] SIDWWHLIYPRP(1)[NT] SWNHWLYSGART(1)[NT] SLFRRRTSTPHR(1)[NT] SWWPFPPQPDPA(1)[NT] NTNPAWRHYKAF(1)[NT] SDNVHTWQAMFK(1)[NT] AYYKTSFPPSTQ(1)[NT] 11 Phage display (common panning) 5 PMID:10219262 CSF samples from neurocysticercosis patient #2 Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA In phage ELISA, the absorbance at 450 nm was measured. Data shown are expressed as the difference between the average A405nm values for selected phage and nonrelated phage G1. NT represents not tested. To increase the specificity of selection, plates coated with anti-human Fc antibody were used in five rounds of biopanning. Sequence similarities between affinity-selected clones and three known T. solium and T. crassiceps proteins were encountered. 1558 VPHIPPN(1) MPPTQVS(1) QMHPWPP(1) QPPFWQF(1) TPPQGLA(1) IPPYNTL(1) AVRPAPL(1) GAKPHPQ(1) QQLSPLP(1) GPPPSPV(1) LPLTPLP(1) 11 Phage display (competitive panning) 3 PMID:11157741 Melanoma-derived growth regulatory protein Not determined. Not determined. Not determined. Ph.D.-7 phage display library (X7) Bound phages were eluted by adding recombinant human MIA at a higher concentration. 1559 QLNVNHQARADQ(1) TSASTRPELHYP(1) TFLPHQMHPWPP(1) VPHIPPNSMALT(1) RLTLLVLIMPAP(5) 5 Phage display (competitive panning) 3 PMID:11157741 Melanoma-derived growth regulatory protein Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) Bound phages were eluted by adding recombinant human MIA at a higher concentration. 1560 SVSVGMKPSPRP(3)[0.313 ± 0.004, 25.0%] YAPSSLLPHSVD(2)[0.236 ± 0.006, 29.2%] NSQLRLNYEKSL(2)[0.494 ± 0.007, 29.5%] MPDHALRPQYSI(1)[0.098 ± 0.006, 13.4%] HLKYATYPPYPQ(1)[0.373 ± 0.010, 35.2%] HNYYNKIQTAAH(1)[0.051 ± 0.008, 16.7%] 6 Phage display (common panning) 3 PMID:11376846 Anti-JEV E protein monoclonal antibody E3.3 Envelope protein Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA ELISA product was determined as A405mAb E3.3 - A405BSA. The ratio of inhibition was determined as 1 - (A405phage)/(A405no phage). M13KO7 phage, wild-type phage acted as negative control. Its absorbance and inhibition ratio were 0.006 ± 0.005 and 11.8%, respectively. The specific peptide ligands presented on ten high-affinity phage clones displayed six different amino acid sequences, all showing a novel cis-proline turn structure. After being superimposed onto the best fit of the three-dimensional structure of JEV E protein, these peptide structures were mapped to a conformational region constituted by three continuous polypeptide segments (E307-E309, E327-E333, E386-E390) in domain III. 1561 SVSVGMKPSPRP(9) 1 Phage display (competitive panning) 8 PMID:11562993 Deoxyribonucleotides, DNA Modification methylase TaqI, M.TaqI Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA In ELISA, the selected phage (SVSVGMKPSPRP) led to an UV absorption increase of 30 mOD/min at 405 nm, while the control phage from the primary library yielded only an increase of 5 mOD/min. For competitive elution using the DNA methyltransferase M.TaqI in the selection step, a biotin-labeled duplex oligodeoxyribonucleotide containing the 5'-TCGA-3' recognition sequence of M.TaqI was employed. Biotinylated DNA-phage complexes were captured with streptavidin-coated paramagnetic beads. The N-terminal amino acid sequence SVSVGMKPSPRP was identified in nine out of ten phages sequenced after eight rounds of selection. 1562 PPPLYF RFCDTS RSRLIW 3 Phage display (common panning) 6 PMID:7980552 Single stranded oligonucleotides consisting of cytosine Not determined. Not determined. Not determined. f3-6mer phage display library (X6) Selection were performed in 50 mM 2[N-Morpholino]ethanesulfonic acid(MES)-buffer. Biotinylated DNA-phage complexes were captured with streptavidin-coated agarose beads. The peptides interacted more strongly with oligo-C compared to the original peptide phage library and wildtype phage. The selected clones also showed different specificity in the interaction with oligo-C, -G, -A and -T. 1563 CSWLHQPYC(13)[23.18 ± 1.16][10.90 ± 0.95] CSWFHMPYC(3)[NT][NT] CSWDHMPYC(2)[NT][NT] CRDYAMPLC(2)[61.87 ± 1.26][36.25 ± 6.85] CKTDPWDYC(2)[NT][NT] CSWVHMPYC(1)[NT][NT] CSWMHMPYC(1)[105.2 ± 1.56][184.36 ± 18.45] CNTIGGYEC(1)[9.64 ± 1.15][23.73 ± 2.09] CLTIGGYEC(1)[NT][NT] CRTLGGYEC(1)[NT][NT] S-W-x-H-[MQ]-P-Y, x-T-[LI]-G(2)-Y-E 10 Phage display (common panning) 3 PMID:11923297 Anti-LOS monoclonal antibody 9-2-L379 L3,7,9 lipooligosaccharide, LOS Not determined. Not determined. Ph.D.-C7C phage display library (CX7C) ELISA,Resonant mirror biosensor The antigenicity of the enriched phage clones against mAb 9-2-L379 was tested by ELISA. Absorbance was measured at 450 nm (data not shown). Phage clones expressing all the cyclic peptides reacted strongly with mAb 9-2-L379. To determine whether the anti-LOS mAb 9-2-L379 interacted with the consensus cyclic peptides by the same paratope as it interacts with LOS, insolution competitive inhibition ELISAs against meningococcal LOS L3,7,9 were performed. Data were shown and expressed as EC50 ± S.E. (μM). No inhibition (NI) was observed for P-CRC, which is a negative clone. Besides, real time binding kinetics between mAb 9-2-L379 and cyclic peptides were measured with a resonant mirror biosensor. The binding affinity is expressed as the equilibrium dissociation constant (KD, nM), calculated from the ratio of Kd/Ka. The KD for LOS is 7.52 ± 3.8 nM. 1564 CSWVHMPYC(1) 1 Phage display (common panning) 4 PMID:11923297 Anti-LOS monoclonal antibody 9-2-L379 L3,7,9 lipooligosaccharide, LOS Not determined. Not determined. Ph.D.-C7C phage display library (CX7C) The fourth round of panning was performed at 50 μg/ml of the target antibody 9-2-L379 coating the microtiter plate wells. 1565 CSWLHMPYC(4)[26.12 ± 1.12][18.76 ± 2.63] CSWDHNPYC(2)[NT][NT] 2 Phage display (common panning) 4 PMID:11923297 Anti-LOS monoclonal antibody 9-2-L379 L3,7,9 lipooligosaccharide, LOS Not determined. Not determined. Ph.D.-C7C phage display library (CX7C) ELISA,Resonant mirror biosensor The antigenicity of the enriched phage clones against mAb 9-2-L379 was tested by ELISA. Absorbance was measured at 450 nm (data not shown). Phage clones expressing all the cyclic peptides reacted strongly with mAb 9-2-L379. To determine whether the anti-LOS mAb 9-2-L379 interacted with the consensus cyclic peptides by the same paratope as it interacts with LOS, insolution competitive inhibition ELISAs against meningococcal LOS L3,7,9 were performed. Data were shown and expressed as EC50 ± S.E. (μM). No inhibition (NI) was observed for P-CRC, which is a negative clone. Besides, real time binding kinetics between mAb 9-2-L379 and cyclic peptides were measured with a resonant mirror biosensor. The binding affinity is expressed as the equilibrium dissociation constant (KD, nM), calculated from the ratio of Kd/Ka. The KD for LOS is 7.52 ± 3.8 nM. The fourth round of panning was performed at 5 μg/ml of the target antibody 9-2-L379 coating the microtiter plate wells. 1566 CSWLHQPYC(18)[23.18 ± 1.16][10.90 ± 0.95] CSWMHMPYC(4)[105.2 ± 1.56][184.36 ± 18.45] CSWFHMPYC(3)[NT][NT] CNTIGGYEC(2)[9.64 ± 1.15][23.73 ± 2.09] S-W-x-H-[MQ]-P-Y, x-T-[LI]-G(2)-Y-E 4 Phage display (common panning) 4 PMID:11923297 Anti-LOS monoclonal antibody 9-2-L379 L3,7,9 lipooligosaccharide, LOS Not determined. Not determined. Ph.D.-C7C phage display library (CX7C) ELISA,Resonant mirror biosensor The antigenicity of the enriched phage clones against mAb 9-2-L379 was tested by ELISA. Absorbance was measured at 450 nm (data not shown). Phage clones expressing all the cyclic peptides reacted strongly with mAb 9-2-L379. To determine whether the anti-LOS mAb 9-2-L379 interacted with the consensus cyclic peptides by the same paratope as it interacts with LOS, insolution competitive inhibition ELISAs against meningococcal LOS L3,7,9 were performed. Data were shown and expressed as EC50 ± S.E. (μM). No inhibition (NI) was observed for P-CRC, which is a negative clone. Besides, real time binding kinetics between mAb 9-2-L379 and cyclic peptides were measured with a resonant mirror biosensor. The binding affinity is expressed as the equilibrium dissociation constant (KD, nM), calculated from the ratio of Kd/Ka. The KD for LOS is 7.52 ± 3.8 nM. The fourth round of panning was performed at 0.5 μg/ml of the target antibody 9-2-L379 coating the microtiter plate wells. 1567 SEPVAML(9) LPPNPTK(7) NLPRLYE(3) TYWYMTP(2) 4 Phage display (common panning) 4 PMID:11923297 Anti-LOS monoclonal antibody 9-2-L379 L3,7,9 lipooligosaccharide, LOS Not determined. Not determined. Ph.D.-7 phage display library (X7) ELISA The antigenicity of the enriched phage clones against mAb 9-2-L379 was tested by ELISA. Absorbance was measured at 450 nm (data not shown). Phage clones expressing the identified linear 7-mer consensus peptides showed no reactivity with mAb 9-2-L379. The fourth round of panning was performed at 50 μg/ml of the target antibody 9-2-L379 coating the microtiter plate wells. 1568 GSMSPYVRWYTP(9) KDASSFQMRPLS(2) SVSVGMKPSPRP(2) KAQSPWSNVDAW(2) HSLKHTQMSYSS(2) 5 Phage display (common panning) 4 PMID:11923297 Anti-LOS monoclonal antibody 9-2-L379 L3,7,9 lipooligosaccharide, LOS Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA The antigenicity of the enriched phage clones against mAb 9-2-L379 was tested by ELISA. Absorbance was measured at 450 nm (data not shown). Phage clones expressing the identified linear 12-mer consensus peptides showed no reactivity with mAb 9-2-L379. The fourth round of panning was performed at 50 μg/ml of the target antibody 9-2-L379 coating the microtiter plate wells. 1569 KVWFLPEAAQPS(1) KVWQMYWPSGQP(1) TSSALKCCFIQ(1) 3 Phage display (subtractive panning) 3 PMID:15755589 Anti-HIV-1 monoclonal antibdoy 2G12 Human immunodeficiency virus type 1, HIV-1 Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA In ELISA, phage clones (displaying KVWFLPEAAQPS, KVWQMYWPSGQP and TSSALKCCFIQ, respectively) bound to 2G12 but not to human IgG. For each round of panning, the library was preadsorbed on BSA. 1570 KCCYYDHSHALS(3) QPTSHRDLRPPI(2) HSLKNSMLTVMA(2) HLHVHLSLSRPL(2) KVWDIRTADNLH(1) KVWDIRYTTPHA(1) EARVFSSKHWIP(1) TMKCCYSNTSPP(1) HLHVHVKFADRQ(1) 9 Phage display (subtractive panning) 5 PMID:15755589 Anti-HIV-1 monoclonal antibdoy 2G12 Human immunodeficiency virus type 1, HIV-1 Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA,Competition experiment In ELISA, phage clones bound to 2G12 but not to human IgG. Besides, the participation of carbohydrate binding sites in the peptide binding was demonstrated by inhibition of the binding of 2G12 to phages (KCCYYDHSHALS, KVWDIRYTTPHA and QPTSHRDLRPPI) or HIV gp160 by mannose, fructose, glucose, mannan and α-D methylmannopyranoside. The binding was specifically inhibited by the mannose containing carbohydrates as well as by fructose but not by glucose. For the first three rounds of panning, the library was preadsorbed on BSA and panned on 2G12. Peptides were selected after adsorbing the library selected in the previous step on human IgG and panning once more on 2G12 in the presence of human IgG (rounds 4 and 5). 1571 SVSVGMKPSPRP(6) KLWQLFPPSAVS(1) EARVXSSKHWIP(1) 3 Phage display (subtractive panning, competitive panning) 6 PMID:15755589 Concanavalin-A, Con A Methyl α-D-mannopyranoside Not determined. Not determined. Ph.D.-12 phage display library (X12) For the first three rounds of panning, the library was preadsorbed on BSA and panned on 2G12. The amplified eluent from the third round of panning was adsorbed on human Immunoglobulin G (huIgG). The non-binding phage was used as input for a fifth round of panning on 2G12 carried out in the presence of huIgG in the liquid phase. Peptides were selected after the library from round 5 was panned on Con A and the bound phages eluted by α-D-methylmannopyranoside. 1572 TMGFTAPREPHY(3) IERPLHESVLAT(2) NNYDDISLRARP(2) AIPNKLNVWPPH(1) TGVSWSVAQPSF(1) SQELTQRPYKWH(1) TPSYINLXDFIA(1) GTSTFNSVPVRD(1) KLTFLNYAEVLR(1) 9 Phage display (subtractive panning, competitive panning) 4 PMID:16603084 Hsp70-peptide complexes, Hsp70-PCs Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) The blocking, binding and washing strategies were carried out as instructed by the manufacturer with the following exceptions. Authors used (a) either 1% Bovine Serum Albumin (BSA) or casein for blocking nonspecific binding alternating these blocking substrates between subsequent rounds of bio-panning to prevent selection of phage recognising the blocking substances, (b) competitive elution with the bait Hsp70-PCs in rounds three and four and (c) bio-panning in solution for rounds two and four, using biotinylated Hsp70-PCs and a streptavidin matrix to prevent selection of plastic-binding phages. In the latter case, 10 μg of Hsp70-PCs were biotinylated using NHS-Biotin according to the manufacturer\'s instructions and incubated with phage particles as recommended by the manufacturer with either of the blocking reagents. The Hsp70-PCs bound phage particles were recovered either through a Streptavidin-agarose (Pierce Chemical Co. ILL) column or Streptavidin-Dynabeads (Dynal Co., Norway) followed by washing and competitive elution as described above. Following each round of bio-panning, the eluted phages were amplified to high titer according to supplier\'s instructions. A subtraction screening using the peptide depleted Hsp70-PC was performed after the third biopanning to remove those phages recognising the Hsp70 portion of the Hsp70-PC bait. The unbound fraction was amplified and used in the fourth round bio-panning. The phage particles from the final fourth round eluate were plated at low density to allow isolation of single phage clones. Three separate bio-panning experiments were performed using Hsp70-PCs as bait. In each case, approximately 400-1000 phages were retained after four rounds of panning. A total of twenty four phage clones were selected at random for further analysis. 1573 SVSVGMKPSPRP(0.46) FHSDWPGXTLTW(0.18) LHAETRSAMHRT(0.09) WKHTSQPPRLIF(0.09) KAXTPVQSASNV(0.09) RTHDNSWNYTSS(0.09) 6 Phage display (subtractive panning, competitive panning) 4 PMID:16603084 Peptide NNYDDISLRARP Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) The blocking, binding and washing strategies were carried out as instructed by the manufacturer with the following exceptions. Authors used (a) either 1% Bovine Serum Albumin (BSA) or casein for blocking nonspecific binding alternating these blocking substrates between subsequent rounds of bio-panning to prevent selection of phage recognising the blocking substances, (b) competitive elution with the bait peptide in rounds three and four and (c) bio-panning in solution for rounds two and four, using biotinylated peptide and a streptavidin matrix to prevent selection of plastic-binding phages. In the latter case, 10 μg of peptide were biotinylated using NHS-Biotin according to the manufacturer\'s instructions and incubated with phage particles as recommended by the manufacturer with either of the blocking reagents. The peptide bound phage particles were recovered either through a Streptavidin-agarose (Pierce Chemical Co. ILL) column or Streptavidin-Dynabeads (Dynal Co., Norway) followed by washing and competitive elution as described above. Following each round of bio-panning, the eluted phages were amplified to high titer according to supplier\'s instructions. The amplified fraction was used in the fourth round biopanning. The phage particles from the final fourth round eluate were plated at low density to allow isolation of single phage clones. 1574 DSPQNPKTWKYI(1.00) 1 Phage display (subtractive panning, competitive panning) 4 PMID:16603084 Peptide TMGFTAPREPHY Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA The specificity of the interaction between the TMGFTAPREPHY and DSPQNPKTWKYI peptides was examined by ELISA. Phages displaying the DSP peptide specifically bind to a synthetic biotinylated TMG peptide. Conversely, phages displaying the TMG peptide bind to a synthetic biotinylated DSP peptide. The blocking, binding and washing strategies were carried out as instructed by the manufacturer with the following exceptions. Authors used (a) either 1% Bovine Serum Albumin (BSA) or casein for blocking nonspecific binding alternating these blocking substrates between subsequent rounds of bio-panning to prevent selection of phage recognising the blocking substances, (b) competitive elution with the bait peptide in rounds three and four and (c) bio-panning in solution for rounds two and four, using biotinylated peptide and a streptavidin matrix to prevent selection of plastic-binding phages. In the latter case, 10 μg of peptide were biotinylated using NHS-Biotin according to the manufacturer\'s instructions and incubated with phage particles as recommended by the manufacturer with either of the blocking reagents. The peptide bound phage particles were recovered either through a Streptavidin-agarose (Pierce Chemical Co. ILL) column or Streptavidin-Dynabeads (Dynal Co., Norway) followed by washing and competitive elution as described above. Following each round of bio-panning, the eluted phages were amplified to high titer according to supplier\'s instructions. The amplified fraction was used in the fourth round biopanning. The phage particles from the final fourth round eluate were plated at low density to allow isolation of single phage clones. 1575 SVSVGMKPSPRP(0.32) GLPPYSPHRLAQ(0.15) NFMESLPRLGMH(0.15) NAQNYSQQAPRP(0.15) HGLHQMSGNTKR(0.15) HPHQPIERQTVQ(0.08) 6 Phage display (subtractive panning, competitive panning) 4 PMID:16603084 Peptide IERPLHESVLAT Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) The blocking, binding and washing strategies were carried out as instructed by the manufacturer with the following exceptions. Authors used (a) either 1% Bovine Serum Albumin (BSA) or casein for blocking nonspecific binding alternating these blocking substrates between subsequent rounds of bio-panning to prevent selection of phage recognising the blocking substances, (b) competitive elution with the bait peptide in rounds three and four and (c) bio-panning in solution for rounds two and four, using biotinylated peptide and a streptavidin matrix to prevent selection of plastic-binding phages. In the latter case, 10 μg of peptide were biotinylated using NHS-Biotin according to the manufacturer\'s instructions and incubated with phage particles as recommended by the manufacturer with either of the blocking reagents. The peptide bound phage particles were recovered either through a Streptavidin-agarose (Pierce Chemical Co. ILL) column or Streptavidin-Dynabeads (Dynal Co., Norway) followed by washing and competitive elution as described above. Following each round of bio-panning, the eluted phages were amplified to high titer according to supplier\'s instructions. The amplified fraction was used in the fourth round biopanning. The phage particles from the final fourth round eluate were plated at low density to allow isolation of single phage clones.