research article phage display screening for tumor necrosis...

Hindawi Publishing CorporationInternational Journal of PeptidesVolume 2013 Article ID 348409 9 pageshttpdxdoiorg1011552013348409

Research ArticlePhage Display Screening forTumor Necrosis Factor-120572-Binding Peptides Detection ofInflammation in a Mouse Model of Hepatitis

Coralie Sclavons1 Carmen Burtea1 Seacutebastien Boutry2 Sophie Laurent1

Luce Vander Elst1 and Robert N Muller12

1 Department of General Organic and Biomedical Chemistry NMR and Molecular Imaging Laboratory University of MonsMendeleıev Building 19 Avenue Maistriau 7000 Mons Belgium

2Center for Microscopy and Molecular Imaging (CMMI) 8 Rue Adrienne Bolland 6041 Gosselies Belgium

Correspondence should be addressed to Robert N Muller robertmullerumonsacbe

Received 19 October 2012 Accepted 3 January 2013

Academic Editor Tzi Bun Ng

Copyright copy 2013 Coralie Sclavons et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

TNF-120572 is one of the most abundant cytokines produced in many inflammatory and autoimmune conditions such as multiplesclerosis chronic hepatitis C or neurodegenerative diseases These pathologies remain difficult to diagnose and consequentlydifficult to treat The aim of this work is to offer a new diagnostic tool by seeking new molecular probes for medical imagingThe target-specific part of the probe consists here of heptameric peptides selected by the phage display technology for their affinityfor TNF-120572 Several affinity tests allowed isolating 2 peptides that showed the best binding capacity to TNF-120572 Finally the bestpeptide was synthesized in both linear and cyclic forms and tested on the histological sections of concanavalin-A-(ConA-)treatedmice liver In this well-known hepatitis mouse model the best results were obtained with the cyclic form of peptide 2 whichallowed for the staining of inflamed areas in the liver The cyclic form of peptide 2 (2C) was thus covalently linked to iron oxidenanoparticles (magnetic resonance imaging (MRI) contrast agent) and tested in the ConA-induced hepatitis mouse model Thevectorized nanoparticles allowed for the detection of inflammation as well as of the free peptide These ex vivo results suggest thatphage display-selected peptides can direct imaging contrast agents to inflammatory areas

1 Introduction

Tumor necrosis factor alpha (TNF-120572) is a proinflammatorycytokine produced in many inflammatory and autoimmunediseases such as rheumatoid arthritis Crohnrsquos disease multi-ple sclerosis or chronic hepatitis C [1 2] TNF-120572 is producedby different cell types including macrophages monocytes T-cells smooth muscle cells adipocytes and fibroblasts Thiscytokine is also implicated in the diseases of the centralnervous system like Alzheimerrsquos and Parkinsonrsquos diseases [3]where it can be produced by several cell populations includ-ing microglia astrocytes endothelial cells Th1 lymphocytesand neurons

Mature TNF-120572 is secreted as a 157-amino acid form [4]with a molecular weight of 17 kDa [5] Before being released

from cells TNF-120572 is anchored in the plasma membraneas a 26 kDa precursor containing both hydrophobic andhydrophilic regions [6] The 17 kDa form of TNF-120572 is excisedfrom the integral transmembrane precursor by proteolyticcleavagemediated by the tumornecrosis factor alpha convert-ing enzyme (TACE) [7] Soluble and transmembrane TNF-120572are produced by cells as homotrimers that bind to two kindsof receptors TNF-RI and TNF-RII (tumor necrosis factorreceptor type I p55 type II p75 resp) which are present inthe membrane of all cell types except erythrocytes

TNF-120572 which is one of the most abundant cytokinesinvolved in apoptotic and inflammatory pathways has bothdesirable and undesirable effects Tumor necrosis inductionand mediation of the immune response to bacterial viraland parasitic invasions are beneficial functions of TNF-120572

2 International Journal of Peptides

[6] It is also an acute phase protein that initiates a cascadeof cytokines and increases vascular permeability therebyrecruiting macrophages and neutrophils to a site of infectionHowever TNF-120572 can also have pathological consequencessuch as promoting the growth of some tumor cell types Italso plays an important role in the chronic inflammation thatoccurs in various pathologies and has been identified as themajor mediator in various autoimmune diseases [8 9] TNF-120572 thus represents a good marker of inflammatory events

Phage display is a high-throughput screening (HTS)method It is an effective way of selecting target-specificproteins and peptides that can be synthesized and linked toan imaging reporter for diagnostic useThis technique can beused to identify peptides or antibodies capable of interactingwith inflammatory mediators [10 11]

In the present work a heptapeptide phage display librarywas screened against TNF-120572 as a first step in the developmentof newmolecular imaging tools for detecting inflammation inchronic inflammatory pathologies and diseases of uncertaindiagnosis Peptide specificity for the cytokine was tested onhistological liver sections of mice treated with concanavalinA (ConA) which is a mitogenic lectin known to stimulatelymphocytes to produce lymphokines [12] mediating chronicinflammation processes [13] and inducing severe injury tohepatocytes [14] ConA induces an autoimmune hepatitisdue to massive CD4+ lymphocyte activation and infiltra-tion into the liver parenchyma leading to the secretion ofthe proinflammatory cytokines TNF-120572 interferon-120574 (IFN120574)interleukin (IL)-2 IL-6 granulocyte macrophage-colonystimulating factor and IL-1 [15] The phage display-selectedheptapeptide was then grafted to iron oxide nanoparticles(known for their use as MRI contrast agents) This newpeptidewas thus set up as an iron oxide-based probe and usedto detect inflammatory process on histological liver sectionsprior to further in vivoMRI tests

2 Methods

21 Phage Display

211 The Biopanning of PhD-C7C Phage Display Libraryagainst TNF-120572 The well of an ELISA microtiter plate wascoated with 100 120583L of target solution (01mgmL of humanTNF-120572 (GenScript Corporation Piscataway USA) in 01MNaHCO

3buffer pH 86) by overnight incubation at 4∘C

in a humid chamber The next day the target solution wasremoved and replaced by the blocking buffer (Bovine SerumAlbumin 5mgmL 01MNaHCO

3 pH 86 NaN

3002) for

2 hours and finally washed with Tris-buffered saline (TBS)supplemented with 01 Tween-20 (TBS-T 50mMTris-HCl150mM NaCl pH 74) After negative selection on a BSA-coated well the phage library (2 times 1011 phages in 100 120583L ofTBS-T 01 PhD-C7C New England Biolabs Inc WestburgB X Leusden The Netherlands) was incubated during 1hour at 37∘C with the target After rinsing TNF-120572-boundphages were eluted with 02M glycine-HCl buffer (pH 22)complemented with 01 BSA and then neutralized with 1MTris-HCl buffer (pH 91)

Eluted phages were amplified during 4 h30 via Escherichiacoli (ER2738 host strain NewEngland Biolabs Inc) infectionAmplified phages were collected by two precipitations at4∘C in PEG-NaCl solution (20 polyethylene glycol-800025M NaCl) The phage pellet was finally solubilized in aTBS buffer solution (50mMTris-HCl 150mMNaCl pH 75)This succession of steps was repeated 4 times and constitutesa biopanning round The selective pressure was increasedduring the third and the fourth rounds of biopanning byincreasing the Tween-20 concentration in the incubationand rinsing buffers to 03 and 05 respectively andby reducing the incubation time to 45min and 30minrespectively

E coli was grown on a selective medium containingisopropyl-beta-D-thiogalactoside (IPTG) (ICN BiomedicalInc Brussels Belgium) and 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (Xgal) (Sigma-Aldrich BornemBelgium)The phage genome contains a part of the LacZ genethat confers to bacteria the ability to produce120573-galactosidasewhich reacts with the X-gal substrate and results in a blueplaque staining Phage titer is thus achieved by countingphage-infected E coli (blue-colored) colonies after eachbiopanning round

212 Sequencing of Selected Phage Clones The genomesequencing of selected phage clones was based on theSanger method which uses dideoxynucleotides triphos-phate as DNA chain terminators Briefly DNA is extractedby the phenolchloroform extraction procedure [16] anddenatured by several heating cycles Virus genome issequenced by using a Start Mix solution (Beckman Coul-ter Analis Namur Belgium) and a 20-base primer (51015840-CCCTCATAGTTAGCGTAACG-31015840 New England BiolabsInc) located 96 nucleotides upstream to the inserted peptide-encoding sequence The Start Mix solution is the sequencingreaction buffer containing 4 ddNTPs 4 dNTPs and the DNApolymerase enzyme

TheDNA sequencewas analyzed on aCEQ2000XLDNAAnalysis System (Beckman Coulter Analis) The sequencereading was performed automatically using the JaMBW 11software (httpbioinformaticsorgJaMBW)

213 Evaluation of the Affinity of Selected Clones for the Target(a) Estimation of the Apparent Dissociation Constant (119870lowast

119889)

between the Phage Clones and TNF-120572The119870lowast119889of phage clones

against their target was assessed by the ELISA (enzyme-linked immunosorbent assay)method using a range of phagedilutions incubated with the specific target

ELISA plates were treated overnight with the targetsolution (human TNF-120572 001mgmL at 4∘C) The next daythe target solution was replaced by the blocking solutionas described above Plates were then incubated for 2 h at37∘C with serial phage dilutions (65 times 10minus11 ndash 33 times 10minus8M)prepared in PBS containing 03 Tween 20 After rinsing theplate 6 times with the same buffer phages were detected witha peroxidase-conjugated anti-M13 monoclonal antibody(Amersham Pharmacia Biotech Benelux Roosendaal TheNetherlands) diluted 1 5000 in the blocking buffer The

International Journal of Peptides 3

peroxidase detection was performed using 221015840-azino-bis[3-ethylbenztiazoline-6-sulfonic acid diammonium salt](ABTS Sigma-Aldrich 22mg in 100mL of 50mM sodiumcitrate pH 40) supplemented with 005 H

2O2 After

30ndash60min of incubation at room temperature the OD405

values were measured on a microplate reader (Stat-Fax-2100Awareness Technology Inc Fisher Bioblock ScientificTournai Belgium) for 119870lowast

119889evaluation

(b) Competition Experiments with an Anti-TNF-120572 AntibodyCompetition experiments were performed in the same man-ner as the 119870lowast

119889experiments except that clones were set in

competition with an anti-TNF-120572 antibody (RampD SystemsInc Abington UK) in dilutions ranging from 13 times 10minus9 to666 times 10minus7M in a PBS solution Plates were first incubatedfor 30min with 50120583Lwell of antibody dilutions before aone-hour incubation with 50120583L of phage solutions at the119870lowast

119889concentration The IC

50of the anti-TNF-120572 antibody is

the antibody concentration required to inhibit by 50 theinteraction between phages and TNF-120572

214 Synthesis of Biotinylated Peptides Peptides displayedon the surface of selected phage clones were synthesizedin a biotinylated form by NeoMPS (Polypeptide groupStrasbourg France) Biotinylated peptides were synthesizedin two forms a linear form (L) and a cyclic (C) form

215 The Evaluation of the Affinity of Selected Peptides forthe Target The affinity of the selected peptides was evaluatedby ELISA as for the phage clones (see Section 213) Thetarget was immobilized overnight on a 96-well plate at 4∘Cbefore a 2-hour incubationwith a protein-free blocking buffersolution (PFBB Pierce Aalst Belgium) and then with a rangeof peptide dilutions 10acircĂŠ3M to 10acircĂŠ6M The target-boundbiotinylated peptideswere detected usingABC (AvidinBiotinComplex) and ABTS solutions as described above Thedissociation constant was estimated from the inflection pointof the curve

22 The Physicochemical Characterization of the 2C Peptide-Grafted Nanoparticles The phage display-selected 2C pep-tide was grafted to ultrasmall nanoparticles of iron oxide(USPIO) The particles were pegylated with the amino-PEG750 (Fluka Bornem Belgium) to avoid the rapid uptake ofUSPIO by macrophages and to prolong the blood circulationtime

Peptide-USPIO-PEG was prepared in our laboratoryfrom previously described nanoparticles bearing carboxy-lated groups on their surface [17ndash19] USPIOs were function-alized in two successive steps first they were functionalizedwith the peptide and second theywere coupled to an amino-PEG 750 Briefly 19mg of the peptide and 6mg of EDCI (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) were addedto 15mL of nanoparticles ([Fe] = 0175Mol PeptideFe molarpercent is 002) and the mixture was stirred overnightThe nanoparticle suspension was ultrafiltrated on a 30 kDmembrane Amino-PEG 750 (0503 g) and EDCI (0327 g)were added to the nanoparticles (PEGFe molar percent is

2) and the reaction was stirred during 17 hThemixture wasultrafiltrated on a 30 kDmembrane to remove low-molecularmaterial

NMRD (Nuclear Magnetic Resonance Dispersion) pro-files were recorded at 37∘C on a Fast Field Cycling Relax-ometer (Stelar Mede Italy) over a magnetic field range from024mT to 024 TAdditional longitudinal (R

1) and transverse

(R2) relaxation rate measurements at 047 T and 141 T were

obtained on Minispec MQ 20 and MQ 60 spin analyzers(Bruker Karlsruhe Germany) respectively The fitting of theNMRDprofiles by a theoretical relaxationmodel [20] allowedthe determination of the crystal radius (r) and the specificmagnetization (119872

119878)

Hydrodynamic sizemeasurementwas carried out by pho-ton correlation spectroscopy (PCS) on a Zetasizer NanoseriesZEN 3600 (Malvern UK)

Total iron concentration was determined by proton (1H)relaxometry at 20MHz and 310K after microwave digestion(MLS-1200 MEGA MILESTONE Analis Namur Belgium)

23 Animals and Treatments Adult male mice C57BL6JOlaHsd (6 months old Harlan Laboratories BV TheNetherlands) were used

All animals were housed in plastic cages under a 12 hlight12 h dark cycle and had free access to food and waterAmbient temperature was maintained at 25 plusmn 2∘C Ani-mals were maintained and treated in compliance with theguidelines specified by the Belgian Ministry of Trade andAgriculture

Adult mice (119899 = 6) received a single ip injection of20mgKg of ConA [21] a lymphocyte T activator producinginflammation in mice liver

24 Immunohistochemistry

241 Tissue Preparation Mice were sacrificed 2 hours afterintravenous injection of ConA and their livers were fixedfor 24 h in 4 paraformaldehyde After fixation the liverwas dehydrated by several alcohol and butanol soakings andthe organ was subsequently embedded in paraffin accordingto standard procedures Sections of 4-micrometer thicknesswere cut for histological tests

242 TNF-120572 Detection by a Polyclonal Antibody in LiverHistological Sections Liver sections were dewaxed and rehy-drated by several washes in toluene and alcohol Theywere subsequently treated for 5 minutes with 2 H

2O2in

order to block endogenous peroxidases Endogenous biotinsand nonspecific epitopes were blocked successively withan avidinbiotin and casein solution before proceeding toincubate the liver sections overnight with a rabbit anti-mouse TNF-120572 polyclonal antibody (1 100 Abcam ParisFrance)The next day liver sections were incubated for 1 hourwith a biotinylated goat anti-rabbit antibody (Vector LabsBrussels Belgium) Reactive sites were detected with theavidin-biotin peroxidase complex (ABC Vector Labs) andrevealed with DAB (331015840-diaminobenzidine Sigma-AldrichBornem Belgium) and 002 of H

2O2prepared in PBS


Sections were washed in distilled water to stop the reactionCounterstaining of liver tissue was performed with Luxol fastblue before mounting in a permanent medium

243 TNF-120572 Detection by 2C and 2L Peptides in LiverHistological Sections TNF-120572 detection by the phage display-selected 2C and 2L peptides was carried out with thesame protocol as for anti-TNF-120572 polyclonal antibody (seeSection 242) Briefly liver sectionswere incubated overnightwith the biotinylated peptides (20120583M) Reactive sites weredetected with the ABC complex and DAB staining

244 TNF-120572 Detection by 2C Peptide-USPIO-PEG in LiverHistological Sections The selected peptide (2C) was graftedtoUSPIO-PEG in order to produce a vectorizedMRI contrastagent for the specific targeting of injured areasThe specificityof the 2C peptide-USPIO-PEG was tested on liver sectionsSlices were deparaffinated in toluene and rehydrated in alco-hol Sections were then incubated overnight with 30mM of2C peptide-USPIO-PEG at 4∘C before being immersed for 30minutes in Prussian blue reagent (5 potassium ferrocyanidein PBS-HCl 2 solution) for the iron staining Slides werecounterstained with eosin during 8 minutes before mountingin a permanent medium

3 Results

31 Characterization of the Selected Phage Clones

311 TNF-120572 Affinity The screening of a cyclic heptamerpeptide library displayed by the phage coat allowed for theselection of 48 phage clones from round 3 and 50 phageclones from round 4 of the selection-amplification procedureTherefore 98 phages presenting an affinity for the target werecollected

Nineteen clones from round 3 and 23 clones from round4 were chosen for their higher affinity for TNF-120572 and theirlower affinity for BSA coated plates (TNF-120572BSA = 29 plusmn 07[mean value plusmn SEM ] for round 3 clones and TNF-120572 BSA =31 plusmn 05 [mean value plusmn SEM ] for round 4 clones)

Based on these results 28 clones with higher affinity werethus selected for further characterization (Figure 1)

312 Peptide Sequence and Biochemical Parameters of theSelected TNF-120572 Binding Clones The DNA of the 28 phageclones selected for their affinity to the TNF-120572 cytokine wassequenced Fifteen different peptide inserts were identifiedPeptide sequences mainly contained hydrophobic aminoacids (61) that is glycine leucine proline and tryptophanoccupying the first 5 positions in the peptide (Figure 2)Hydrophilic amino acids with a hydroxyl function (ieserine and threonine) are less well represented (39) thanhydrophobic amino acids and are most predominant in thelast two positions

Among the fifteen different peptides that were identifiedfrom the 28 determined peptide sequences the cyclic pep-tides C-GLPWLST-C and C-GPAVYMK-C were found 13and 2 times respectively (Table 1)

005

115

225

335

445

Clones

C2-R

3C1

8-R3

C22-

R3C2

5-R3

C30-

R3C3

3-R3

C40-

R3C4

1-R3

C42-

R3C4

3-R3

C45-

R3C4

6-R3

C47-

R3C4

8-R3

C23-

R4C2

7-R4

C28-

R4C2

9-R4

C32-

R4C3

3-R4

C34-

R4C3

5-R4

C37-

R4C4

0-R4

C46-

R4C4

7-R4

C48-

R4C4

9-R4

TNF-120572

BSA

Figure 1 Affinity of the 28 selected clones for the TNF-120572 cytokineResults are presented as a ratio between the clonesrsquo affinity for TNF-120572 and that for BSA Clones show 4-times higher affinity for TNF-120572than for BSA The phage clones were selected after the biopanningrounds 3 and 4

12345670

10

20

30

40

50

60

70

Arg His LysAspGlu Ser ThrTyrAsnGlnCysMet LeuAla Gly Pro Val Ile Phe Trp

Am

ino

acid

s pos

ition

Freq

uenc

y (

)

Figure 2The analysis of the amino acid frequency in each positionof the heptapeptide sequences The 5 first positions are mainlyoccupied by hydrophobic amino acids while the last two positionsare mainly occupied by hydrophilic amino acids

Two biochemical parameters of these peptides weretheoretically estimated by using the EXPASYProtParam tools(httpwwwexpasychtoolsprotparamhtml)

The predicted half-life is the time required for half ofthe intravenously injected peptide dose to be degraded Thepeptidesrsquo half-life must be long enough to allow specificinteractions between peptides and their targets to takeplace (eg during MRI scans with the peptides linked tocontrast agents) The half-life of peptides ranges between08 and 30 hours The longest predicted half-lives (20ndash30hours) were found for the following peptides C-PATLTSL-C C-GPAVYMK-C C-GLPWLST-C and C-GSKTQAP-C(Table 1)

The isoelectric point (pI) provides information that isimportant for further applications in physiological condi-tions Indeed an electrical charge borne by the peptide atphysiological pH can change its biodistribution in vivo Achange of the peptide charge can also affect the affinity for


Table 1 Biochemical properties of the selected peptide clones (fromhttpwwwexpasychtoolsprotparamhtml)

Clone Sequence T12

(hours) pIC2-R3 C-GSKTQAP-C 30 806C18-R3 C-GLPWLST-C 30 551C22-R3 C-STPHNLG-C 19 672C25-R3 C-LATGNQI-C 55 551C30-R3 C-PATLTSL-C 20 551C33-R3 C-HGAPNRL-C 35 808C41-R3 C-RPPIGAF-C 1 807C42-R3 C-TSQSQHM-C 72 672C45-R3 C-GPAVYMK-C 30 805C46-R3 C-QGDLPGY-C 08 380C32-R4 C-SPHTTIA-C 19 672C33-R4 C-EPFAGRS-C 1 599C35-R4 C-TSPLPGT-C 72 551C46-R4 C-SYEAHQT-C 19 524C47-R4 C-NNPLKSL-C 14 806T12 half-life theoretically estimated inmammalian reticulocytes in vitro andbased on the N-end rule [20]pI isoelectric point which is the pH at which a particularmolecule or surfacecarries no net electrical charge

the target and consequently that of the conjugated imagingagentThe pI values of the 28 selected peptides were found tovary from 38 to 808 Eight of them would be in an ionizedstate at a pH near the physiological pH of 74 (Table 1 boldtext)

313 Estimation of the119870lowast119889between the Phage Clones and TNF-

120572 The119870lowast119889value was estimated for 15 phage clones One clone

was chosen to represent the sequences thatwere found severaltimes The clone that showed the best 119870lowast

119889value is C30-R3

(clone 30 from round 3 C-PATLTSL-C119870lowast119889= 205times 10minus11M)

The lowest affinity was found for cloneC47-R4 (clone 47 fromround 4 C-NNPLKSL-C 119870lowast

119889= 989 times 10minus6M) All the other

clones were found to have a119870lowast119889value lower than 10minus8M

The best clones were chosen on the basis of the ratio 119870lowast119889

BSA119870lowast119889TNF-120572 to be tested in competition with an anti-

TNF-120572 antibody (Figure 3) Seven phage clones were thusselected for these competition tests C2-R3 (C-GSKTQAP-C) C22-R3 (C-STPHNLG-C) C30-R3 (C-PATLTSL-C) C41-R3 (C-RPPIGAF-C) C42-R3 (C-TSQSQHM-C) C45-R3 (C-GPAVYMK-C) and C46-R3 (C-QGDLPGY-C) The high-est affinity clones were C2-R3 and C30-R3 with a 119870

119889

BSA119870119889TNF-120572 ratio of 6 times 106 and 42 times 106 respectively

the clone showing the lowest affinity was again C47-R4 witha 119870119889BSA119870

119889TNF-120572 ratio of 002

314 Competition Experiments with an Anti-TNF-120572AntibodyThe objective of this test is to set up a competition betweenevery phage clone and another ligand of the target tocalculate an inhibition constant value (IC

50) A high IC

50

value indicates that a significant concentration of antibody is

Clones

C2-R

3C1

8-R3

C22-

R3C2

5-R3

C30-

R3C3

3-R3

C41-

R3C4

2-R3

C45-

R3C4

6-R3

C32-

R4C3

3-R4

C35-

R4C4

6-R4

C47-

R4

1119864minus021119864minus011119864+001119864+011119864+021119864+031119864+041119864+051119864+061119864+07

119870119889

BSA

119870119889

TNF-120572

Figure 3 Affinity of the 15 selected phage clones Clones having thebest affinity are the C2-R3 and C30-R3 clones with a 119870

119889BSA119870

119889

TNF-120572 of 6 times 106 and 42 times 106 respectively

necessary to displace phages from their binding sites on thetarget

The best phage clone-target interaction was found for C2-R3 andC30-R3 (IC

50values of 112times 10minus7Mand 127times 10minus7M

resp)Subsequently the best phage clones were selected on the

basis of their ICAb anti-TNF-12057250

119870clone119889

ratio which reflects thehighest target affinity of a phage clone (Figure 4) The clonesC2-R3 and C30-R3 showed the highest ratio (1087 and 6195resp)

32 The Characterization of the PhageDisplay-Selected Peptides

321 The Estimation of 119870lowast119889of the Selected Peptides for TNF-

120572 The 119870lowast119889values of cyclic and linear biotinylated peptides

(C-)GSKTQAP(-C) (peptide 1) and (C-)PATLTSL(-C) (pep-tide 2) were evaluated (C-)PATLTSL(-C) showed a betteraffinity for the target than (C-)GSKTQAP(-C) Linear andcyclic forms of GSKTQAP weakly interact with TNF-120572 andthe curve does not reach saturation In comparison withthe GSKTQAP peptide the linear and cyclic forms of thePATLTSL peptide (resp 2L and 2C peptides) have a goodability to interact with the target (119870lowast

119889values of 179times10minus4 and

112 times 10minus4 resp) Both linear and cyclic forms show only a

weak interaction with the PFBB coated plate the 119870lowast119889values

could not be estimated

33 The Physicochemical Characterization of the 2C PeptideGrafted to the USPIO-PEG Nanoparticles The 2C peptide-USPIO-PEG was prepared from iron oxide nanoparticlesthat were functionalized first with the peptide and thenwith an amino-PEG 750 These grafting steps do not changesignificantly the relaxometric properties of the nanoparticleNMRD profiles showing the changes in relaxivity (1T1)versus the applied magnetic field strength are given inFigure 5

The physicochemical properties of the nanoparticlesincluded a hydrodynamic size of 33 nm (determined by PCS)


108738

7202

619512

7176

7261

16528

25984

0 1000 2000 3000 4000 5000 6000

C2-R3 (C-GSKTQAP-C)

C22-R3 (C-STPHNLG-C)

C30-R3 (C-PATLTSL-C)

C41-R3 (C-RPPIGAF-C)

C42-R3 (C-TSQSQHM-C)

C45-R3 (C-GPAVYMK-C)

C46-R3 (C-QGDLPGY-C)

ICAC anti-TNF12057250

Kcloned

Figure 4 ICAC anti-TNF-12057250

119870clone119889

ratio of the 7 phage clones selected for their high 119870119889BSA119870

119889TNF-120572 ratio Clones C2-R3 and C30-R3 have

the best affinity for the target (ICAC anti-TNF-12057250

119870clone119889

ratio values of 1087 and 6195 resp) These results are in agreement with the previousones

Protonic Larmor frequency (MHz)001 01 1 10 100 1000

0

10

20

30

40

50

60

70

Rela

xivi

ty(sminus1

m

Mminus1)

Figure 5 NMRD profiles of 2C peptide-USPIO-PEG (black circles)and USPIO-PEG (red triangles)

an 1199031of 402 sminus1mMminus1 and 119903

2of 816 sminus1mMminus1 at 20MHz

37∘C an 1199031of 179 sminus1mMminus1 and 119903

2of 829 sminus1mMminus1 at

60MHz 37∘CThe theoretical fitting of the NMRD profile according

to the superparamagnetic relaxation model [20] gave amagnetizationMsat of 5582Am2kg and a radius of 576 nmThe vectorization with peptides and PEG does not changesignificantly the magnetic properties of the nanoparticles

34 Immunohistochemical Assay

341 TNF-120572 Immunodetection with a Polyclonal Antibody in aMouse Model of Hepatitis To confirm the specific affinity of

the phage display-selected peptide PATLTSL called peptide2 to TNF-120572 the proinflammatory cytokine was targeted in awell-known mouse model of hepatitis

TNF-120572 was detected with a polyclonal anti-TNF-120572 anti-body on liver tissue sections (Figures 6(a) and 6(b)) and wasmainly detected around hepatic sinusoids 2 hours after ivConA injection

342 TNF-120572 Detection by the Phage Display-Selected 2C and2L Peptides The affinity of the phage display-selected 2C and2L peptides for TNF-120572 was evaluated by immunohistochem-istry on liver sections obtained frommice treated with ConAThe cyclic form of peptide 2 produced a similar stainingto that obtained with the polyclonal anti-TNF-120572 antibody(Figures 6(c) and 6(d)) On the contrary the linear form didnot yield such a result

343 TNF-120572 Detection with the Phage Display-Selected 2CPeptide Grafted to USPIO-PEG After proving the affinityof the biotinylated 2C peptide on liver sections this phagedisplay-selected peptide was grafted to USPIO-PEG to studythe affinity of the vectorized probe for the target USPIOwas detected with the Prussian blue iron staining methodA blue coloration indicated the presence of iron aroundsinusoids and blood vessels on ConA-treated liver sections(Figure 6(e)) similar to the results obtained with the biotiny-lated form of the 2C peptide Healthy liver did not show anyblue coloration (Figure 6(f)) suggesting that the vectorizednanoparticles were able to bind their target in the liversections of ConA-treated mice The selected peptide did notlose its affinity for TNF-120572 after grafting to USPIO

4 Discussion

Inflammation is the first response of the immune systemto infection injury or irritation During the acute phaseof the inflammatory process some molecular and cellular


(a) (b)

(c) (d)

(e) (f)

Figure 6 TNF-120572 detection in liver sections of ConA-treated mice TNF-120572 was first detected with a polyclonal anti-TNF-120572 antibody in liversections of ConA-treated mice by a brown coloration mainly around sinusoids and blood vessels (a) as compared to untreated-liver sections(b) TNF-120572 was then detected with the biotinylated 2C peptide (c) by a similar brown coloration located around sinusoids and blood vesselson liver sections of treated mice The sections of healthy liver did not show such a staining (d) The 2C peptide-USPIO-PEG also allowed forthe detection of the TNF-120572 cytokine on liver sections of treated mice (e) while it was not detectable on healthy liver sections (f) Scale bar100120583m

changes occur like the accumulation of fluid inflamma-tory cells and soluble mediators Many of these solublemediators regulate the activation of resident cells such asmonocytes lymphocytes or neutrophils which results inthe systemic response to the inflammatory process These

mediators include cytokines inflammatory lipid metabolitesarachidonic acid derivatives (prostaglandin leukotrienes)vasoactive amines like histamine or serotonin and cascadesof soluble proteasessubstrates (clotting complement andkinin pathways) which generate numerous proinflammatory


peptides Among these factors cytokines play key roles inmediating inflammatory reactions especially IL and TNFwhich are potent and primary inflammatorymoleculesmedi-ating acute inflammation

TNF-120572 is the most common proinflammatory cytokinesecreted during the inflammatory process It is involved inboth acute and chronic inflammation and has a key positionin the pathogenesis of various infectious and inflammatorydiseases It is abundantly present in inflammatory areas intwo forms a soluble and a transmembrane precursor formsThese characteristics make TNF-120572 a good candidate forinflammation targeting

In the present studywe used the phage display technologyto identify small peptide vectors forMRI detection of inflam-mation

Results showed two phage displayed peptides having abetter affinity for the target (TNF-120572) among a selectionof 28 phage clones To optimize peptide target specificityphage displayed peptides were incubated with TNF-120572 withan increasing concentration of destabilizing detergent duringeach round of selection A competition test with an anti-TNF-120572 antibody was also performed to validate the specificinteraction between peptides and their target

Finally the two best peptides were synthesized in a linearand a cyclic form and 119870lowast

119889values were evaluated The lowest

119870lowast

119889values of free peptides compared to phage displayed

peptides might be explained by a multivalency effect allowedby the five peptide copies displayed by phages [22 23]However it has been reported that a conformation constraintinduced by disulfide cyclization of peptides could attenuatethis affinity-decreasing effect [24]The biotinylated linear andcyclic forms of peptide 2 were then chosen for histologicalstudies on a well-known mouse model of hepatitis TNF-120572was mainly detected around hepatic sinusoids 2 hours afteriv ConA injection in accordance with previously reporteddata [12 21]

The best result was obtained with the cyclic form ofpeptide 2 (called 2C peptide) which allowed detecting TNF-120572as efficiently as the anti-TNF-120572 antibody on liver histologicalsections The cyclization gives the peptide a spatial confor-mation that may facilitate its interaction with the target thusallowing better TNF-120572 staining results than those obtainedwith the linear form of the same peptide

Finally the 2C peptide was grafted to iron oxide nanopar-ticles that were evaluated on liver sections Results obtainedwith the new specific iron oxide nanoparticles were comparedto anti-TNF-120572 antibody and to the synthesized biotinylated2C peptide experiments performed on the ConA-treatedmice liver sections The specific 2C peptide-USPIO-PEGshowed the same labeling as the antibody and the biotinylated2C peptide on histological liver sections showing that theUSPIO-PEG thanks to the TNF-120572 specific 2C peptide linkedto their surface allowed for the staining of this proinflamma-tory cytokine in a ConA-induced mouse model of hepatitis

At present some inflammatory diseases like arthritisasthma atherosclerosis Crohnrsquos disease or neurodegenera-tive diseases are not well understood and consequently notwell diagnosed Molecular imaging which uses small probes(eg MRI contrast agents) vectorized to an injured area

offers new tools for diagnosing idiopathic diseases [25ndash27]Increasingly studies are focusing on new targeted approachesto the detection and treatment of inflammation [28ndash30]

The aim of this work was thus to develop specific ironoxide probes by vectorization with phage display-selectedheptapeptides to offer a new tool for the MRI diagnosisof pathologies with an abundant inflammatory process Inthis molecular approach of MRI magnetic nanoparticlesare optimized to reach a specific target known to be acharacteristic and abundant molecular feature of a certaindisease In this work TNF-120572 was chosen as a target to detectinflammationThe phage display-selected 2C peptide and thespecific 2C peptide-USPIO-PEG seemed to be able to detectinflammation by interacting with TNF-120572 in ConA-treatedmice liver sections

According to these results the 2C peptide is appealing formolecular imaging applications It can for instance be usedas a vector for magnetic probes like iron oxide nanoparticlesor gadolinium complexes providing a new targeted agent forthe detection of inflammation in MRI

Acknowledgments

The authors thank Mrs Patricia de Francisco for her helpin preparing the paper and the following ContractGrantsponsors the European Cooperation in the Field of Sci-entific and Technical Research (COST Action D38) theEuropean Network for Cell Imaging and Tracking Expertise(ENCITE) (Seventh Framework Program) FP7-HEALTH-2007-A Action de Recherches Concertees of the FrenchCommunity of Belgium Convention AUWB-2010ndash1015-UMONS-5 Fonds de la Recherche Scientifique (FNRS) theUniversity of Mons the Center for Microscopy and Molec-ular Imaging (CMMI) which is supported by the EuropeanRegional Development Fund and the Walloon Region

References

[1] H Knobler and A Schattner ldquoTNF-120572 chronic hepatitis C anddiabetes a novel triadrdquo QJMed vol 98 no 1 pp 1ndash6 2005

[2] E Larrea N Garcia C Qian M P Civeira and J PrietoldquoTumor necrosis factor 120572 gene expression and the response tointerferon in chronic hepatitis CrdquoHepatology vol 23 no 2 pp210ndash217 1996

[3] P L McGeer and E G McGeer ldquoInflammation and thedegenerative diseases of agingrdquoAnnals of theNewYorkAcademyof Sciences vol 1035 pp 104ndash116 2004

[4] W Fiers ldquoPrecursor structures and structure-function analysisof TNF and lymphotoxinrdquo Immunology Series vol 56 pp 79ndash92 1992

[5] B B Aggarwal B Moffat and R N Harkins ldquoHuman lympho-toxin Production by a lymphoblastoid cell line purificationand initial characterizationrdquo Journal of Biological Chemistry vol259 no 1 pp 686ndash691 1984

[6] J Vilcek and T H Lee ldquoTumor necrosis factor new insightsinto the molecular mechanisms of its multiple actionsrdquo Journalof Biological Chemistry vol 266 no 12 pp 7313ndash7316 1991

[7] R A Black C T Rauch C J Kozlosky et al ldquoA metallopro-teinase disintegrin that releases tumour-necrosis factor-0 fromcellsrdquo Nature vol 385 no 6618 pp 729ndash733 1997


[8] B Beutler I W Milsark and A C Cerami ldquoPassive immuniza-tion against cachectintumor necrosis factor protectsmice fromlethal effect of endotoxinrdquo Science vol 229 no 4716 pp 869ndash871 1985

[9] P Vassalli ldquoThe pathophysiology of tumor necrosis factorsrdquoAnnual Review of Cell and Developmental Biology vol 10 pp411ndash452 1992

[10] T J M Molenaar C C M Appeldoorn S A M De Haas etal ldquoSpecific inhibition of P-selectin-mediated cell adhesion byphage display-derived peptide antagonistsrdquo Blood vol 100 no10 pp 3570ndash3577 2002

[11] C L Chirinos-Rojas M W Steward and C D PartidosldquoA peptidomimetic antagonist of TNF-120572-mediated cytotoxicityidentified from a phage-displayed random peptide libraryrdquoJournal of Immunology vol 161 no 10 pp 5621ndash5626 1998

[12] E Pick J Brostoff J Krejci and J L Turk ldquoInteraction betweenldquosensitized lymphocytesrdquo and antigen in vitro II Mitogen-induced release of skin reactive and macrophage migrationinhibitory factorsrdquoCellular Immunology vol 1 no 1 pp 92ndash1091970

[13] E Pick and J L Turk ldquoThe biological activities of solublelymphocyte productsrdquo Clinical and Experimental Immunologyvol 10 no 1 pp 1ndash23 1972

[14] G Tiefs J Hentschel and A Wendel ldquoA T cell-dependentexperimental liver injury in mice inducible by concanavalin ArdquoJournal of Clinical Investigation vol 90 no 1 pp 196ndash203 1992

[15] A Nonomura M Tanino H Kurumaya G Ohta Y Katoand K Kobayashi ldquoStudies of immune functions of patientswith chronic hepatitisrdquo The Tohoku Journal of ExperimentalMedicine vol 137 no 2 pp 163ndash177

[16] J Sambrook E F Fritsch and TManiatisMolecular Cloning ALaboratoryManual Cold SpringHarbor Laboratory Press NewYork NY USA 1989

[17] M Port C Corot I Raynal and O Rousseaux ldquoNovel com-positions magnetic particles covered with gem-bisphosphonatederivativesrdquo httpwwwpatentlensnetpatentlenspat-entshtmlpatnums=US 20040253181 A1ampreturnTo=quickhtml

[18] V Rerat S Laurent C Burtea et al ldquoUltrasmall particleof iron oxide-RGD peptidomimetic conjugate synthesis andcharacterisationrdquo Bioorganic and Medicinal Chemistry Lettersvol 20 no 6 pp 1861ndash1865 2010

[19] K A Radermacher N Beghein S Boutry et al ldquoIn vivodetection of inflammation using pegylated iron oxide particlestargeted at e-selectin a multimodal approach using mr imagingand epr spectroscopyrdquo Investigative Radiology vol 44 no 7 pp398ndash404 2009

[20] A Roch RNMuller and PGillis ldquoTheory of proton relaxationinduced by superparamagnetic particlesrdquo Journal of ChemicalPhysics vol 110 p 5403 1999

[21] C Trautwein T Rakemann N P Malek J Plumpe G TiegsandM PManns ldquoConcanavalin A-induced liver injury triggershepatocyte proliferationrdquo Journal of Clinical Investigation vol101 no 9 pp 1960ndash1969 1998

[22] M Mammen S K Choi and G M Whitesides ldquoPolyvalentinteractions in biological systems implications for design anduse of multivalent ligands and inhibitorsrdquo Angewandte ChemieInternational Edition vol 37 no 20 pp 2754ndash2794 1998

[23] J E Gestwicki C W Cairo L E Strong K A Oetjen and LL Kiessling ldquoInfluencing receptor-ligand binding mechanismswith multivalent ligand architecturerdquo Journal of the AmericanChemical Society vol 124 no 50 pp 14922ndash14933 2002

[24] L B Giebel R T Cass D L Milligan D C Young R Arzeand C R Johnson ldquoScreening of cyclic peptide phage librariesidentifies ligands that bind streptavidin with high affinitiesrdquoBiochemistry vol 34 no 47 pp 15430ndash15435 1995

[25] A Chopra ldquo99mTc-labeled murine IgMmonoclonal antibodyfanolesomab that targets the CD15 glycoprotein antigenrdquo inMolecular Imaging and Contrast Agent Database (MICAD)National Library of Medicine (US) NCBI Bethesda Md USA2004ndash2011

[26] C Burtea S Laurent E Lancelot et al ldquoPeptidic targetingof phosphatidylserine for the MRI detection of apoptosis inatherosclerotic plaquesrdquoMolecular Pharmaceutics vol 6 no 6pp 1903ndash1919 2009

[27] L Larbanoix C Burtea E Ansciaux et al ldquoDesign andevaluation of a 6-mer amyloid-beta protein derived phagedisplay library for molecular targeting of amyloid plaques inAlzheimerrsquos disease comparison with two cyclic heptapeptidesderived from a randomized phage display libraryrdquo Peptides vol32 no 6 pp 1232ndash1243 2011

[28] M Kopf M F Bachmann and B J Marsland ldquoAvertinginflammation by targeting the cytokine environmentrdquo NatureReviews Drug Discovery vol 9 pp 703ndash718 2010

[29] E Bell ldquoInflammation targeting TNFrdquo Nature ReviewsImmunology vol 9 pp 390ndash391 2009

[30] P Laverman C P Bleeker-Rovers F H M Corstens O CBoerman and W J G Oyen ldquoDevelopment of infection andinflammation targeting compoundsrdquoCurrent Radiopharmaceu-ticals vol 1 no 1 pp 42ndash48 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


[6] It is also an acute phase protein that initiates a cascadeof cytokines and increases vascular permeability therebyrecruiting macrophages and neutrophils to a site of infectionHowever TNF-120572 can also have pathological consequencessuch as promoting the growth of some tumor cell types Italso plays an important role in the chronic inflammation thatoccurs in various pathologies and has been identified as themajor mediator in various autoimmune diseases [8 9] TNF-120572 thus represents a good marker of inflammatory events

Phage display is a high-throughput screening (HTS)method It is an effective way of selecting target-specificproteins and peptides that can be synthesized and linked toan imaging reporter for diagnostic useThis technique can beused to identify peptides or antibodies capable of interactingwith inflammatory mediators [10 11]

In the present work a heptapeptide phage display librarywas screened against TNF-120572 as a first step in the developmentof newmolecular imaging tools for detecting inflammation inchronic inflammatory pathologies and diseases of uncertaindiagnosis Peptide specificity for the cytokine was tested onhistological liver sections of mice treated with concanavalinA (ConA) which is a mitogenic lectin known to stimulatelymphocytes to produce lymphokines [12] mediating chronicinflammation processes [13] and inducing severe injury tohepatocytes [14] ConA induces an autoimmune hepatitisdue to massive CD4+ lymphocyte activation and infiltra-tion into the liver parenchyma leading to the secretion ofthe proinflammatory cytokines TNF-120572 interferon-120574 (IFN120574)interleukin (IL)-2 IL-6 granulocyte macrophage-colonystimulating factor and IL-1 [15] The phage display-selectedheptapeptide was then grafted to iron oxide nanoparticles(known for their use as MRI contrast agents) This newpeptidewas thus set up as an iron oxide-based probe and usedto detect inflammatory process on histological liver sectionsprior to further in vivoMRI tests

2 Methods

21 Phage Display

211 The Biopanning of PhD-C7C Phage Display Libraryagainst TNF-120572 The well of an ELISA microtiter plate wascoated with 100 120583L of target solution (01mgmL of humanTNF-120572 (GenScript Corporation Piscataway USA) in 01MNaHCO

3buffer pH 86) by overnight incubation at 4∘C

in a humid chamber The next day the target solution wasremoved and replaced by the blocking buffer (Bovine SerumAlbumin 5mgmL 01MNaHCO

3 pH 86 NaN

3002) for

2 hours and finally washed with Tris-buffered saline (TBS)supplemented with 01 Tween-20 (TBS-T 50mMTris-HCl150mM NaCl pH 74) After negative selection on a BSA-coated well the phage library (2 times 1011 phages in 100 120583L ofTBS-T 01 PhD-C7C New England Biolabs Inc WestburgB X Leusden The Netherlands) was incubated during 1hour at 37∘C with the target After rinsing TNF-120572-boundphages were eluted with 02M glycine-HCl buffer (pH 22)complemented with 01 BSA and then neutralized with 1MTris-HCl buffer (pH 91)

Eluted phages were amplified during 4 h30 via Escherichiacoli (ER2738 host strain NewEngland Biolabs Inc) infectionAmplified phages were collected by two precipitations at4∘C in PEG-NaCl solution (20 polyethylene glycol-800025M NaCl) The phage pellet was finally solubilized in aTBS buffer solution (50mMTris-HCl 150mMNaCl pH 75)This succession of steps was repeated 4 times and constitutesa biopanning round The selective pressure was increasedduring the third and the fourth rounds of biopanning byincreasing the Tween-20 concentration in the incubationand rinsing buffers to 03 and 05 respectively andby reducing the incubation time to 45min and 30minrespectively

E coli was grown on a selective medium containingisopropyl-beta-D-thiogalactoside (IPTG) (ICN BiomedicalInc Brussels Belgium) and 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (Xgal) (Sigma-Aldrich BornemBelgium)The phage genome contains a part of the LacZ genethat confers to bacteria the ability to produce120573-galactosidasewhich reacts with the X-gal substrate and results in a blueplaque staining Phage titer is thus achieved by countingphage-infected E coli (blue-colored) colonies after eachbiopanning round

212 Sequencing of Selected Phage Clones The genomesequencing of selected phage clones was based on theSanger method which uses dideoxynucleotides triphos-phate as DNA chain terminators Briefly DNA is extractedby the phenolchloroform extraction procedure [16] anddenatured by several heating cycles Virus genome issequenced by using a Start Mix solution (Beckman Coul-ter Analis Namur Belgium) and a 20-base primer (51015840-CCCTCATAGTTAGCGTAACG-31015840 New England BiolabsInc) located 96 nucleotides upstream to the inserted peptide-encoding sequence The Start Mix solution is the sequencingreaction buffer containing 4 ddNTPs 4 dNTPs and the DNApolymerase enzyme

TheDNA sequencewas analyzed on aCEQ2000XLDNAAnalysis System (Beckman Coulter Analis) The sequencereading was performed automatically using the JaMBW 11software (httpbioinformaticsorgJaMBW)

213 Evaluation of the Affinity of Selected Clones for the Target(a) Estimation of the Apparent Dissociation Constant (119870lowast

119889)

between the Phage Clones and TNF-120572The119870lowast119889of phage clones

against their target was assessed by the ELISA (enzyme-linked immunosorbent assay)method using a range of phagedilutions incubated with the specific target

ELISA plates were treated overnight with the targetsolution (human TNF-120572 001mgmL at 4∘C) The next daythe target solution was replaced by the blocking solutionas described above Plates were then incubated for 2 h at37∘C with serial phage dilutions (65 times 10minus11 ndash 33 times 10minus8M)prepared in PBS containing 03 Tween 20 After rinsing theplate 6 times with the same buffer phages were detected witha peroxidase-conjugated anti-M13 monoclonal antibody(Amersham Pharmacia Biotech Benelux Roosendaal TheNetherlands) diluted 1 5000 in the blocking buffer The



2O2 After



119889evaluation













1) and transverse



119878)









2O2in


2O2prepared in PBS





3 Results







005

115

225

335

445

Clones

C2-R

3C1

8-R3

C22-

R3C2

5-R3

C30-

R3C3

3-R3

C40-

R3C4

1-R3

C42-

R3C4

3-R3

C45-

R3C4

6-R3

C47-

R3C4

8-R3

C23-

R4C2

7-R4

C28-

R4C2

9-R4

C32-

R4C3

3-R4

C34-

R4C3

5-R4

C37-

R4C4

0-R4

C46-

R4C4

7-R4

C48-

R4C4

9-R4

TNF-120572

BSA


12345670

10

20

30

40

50

60

70


Am

ino

acid

s pos

ition

Freq

uenc

y (

)







Clone Sequence T12














119889



119889TNF-120572 ratio of 002


50) A high IC

50


Clones

C2-R

3C1

8-R3

C22-

R3C2

5-R3

C30-

R3C3

3-R3

C41-

R3C4

2-R3

C45-

R3C4

6-R3

C32-

R4C3

3-R4

C35-

R4C4

6-R4

C47-

R4

1119864minus021119864minus011119864+001119864+011119864+021119864+031119864+041119864+051119864+061119864+07

119870119889

BSA

119870119889

TNF-120572


119889BSA119870

119889







119870clone119889













108738

7202

619512

7176

7261

16528

25984

0 1000 2000 3000 4000 5000 6000

C2-R3 (C-GSKTQAP-C)








Kcloned


119870clone119889




119870clone119889



0

10

20

30

40

50

60

70

Rela

xivi

ty(sminus1

m

Mminus1)














4 Discussion



(a) (b)

(c) (d)

(e) (f)











119870lowast









Acknowledgments


References







































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



2O2 After



119889evaluation













1) and transverse



119878)









2O2in


2O2prepared in PBS





3 Results







005

115

225

335

445

Clones

C2-R

3C1

8-R3

C22-

R3C2

5-R3

C30-

R3C3

3-R3

C40-

R3C4

1-R3

C42-

R3C4

3-R3

C45-

R3C4

6-R3

C47-

R3C4

8-R3

C23-

R4C2

7-R4

C28-

R4C2

9-R4

C32-

R4C3

3-R4

C34-

R4C3

5-R4

C37-

R4C4

0-R4

C46-

R4C4

7-R4

C48-

R4C4

9-R4

TNF-120572

BSA


12345670

10

20

30

40

50

60

70


Am

ino

acid

s pos

ition

Freq

uenc

y (

)







Clone Sequence T12














119889



119889TNF-120572 ratio of 002


50) A high IC

50


Clones

C2-R

3C1

8-R3

C22-

R3C2

5-R3

C30-

R3C3

3-R3

C41-

R3C4

2-R3

C45-

R3C4

6-R3

C32-

R4C3

3-R4

C35-

R4C4

6-R4

C47-

R4

1119864minus021119864minus011119864+001119864+011119864+021119864+031119864+041119864+051119864+061119864+07

119870119889

BSA

119870119889

TNF-120572


119889BSA119870

119889







119870clone119889













108738

7202

619512

7176

7261

16528

25984

0 1000 2000 3000 4000 5000 6000

C2-R3 (C-GSKTQAP-C)








Kcloned


119870clone119889




119870clone119889



0

10

20

30

40

50

60

70

Rela

xivi

ty(sminus1

m

Mminus1)














4 Discussion



(a) (b)

(c) (d)

(e) (f)











119870lowast









Acknowledgments


References







































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





3 Results







005

115

225

335

445

Clones

C2-R

3C1

8-R3

C22-

R3C2

5-R3

C30-

R3C3

3-R3

C40-

R3C4

1-R3

C42-

R3C4

3-R3

C45-

R3C4

6-R3

C47-

R3C4

8-R3

C23-

R4C2

7-R4

C28-

R4C2

9-R4

C32-

R4C3

3-R4

C34-

R4C3

5-R4

C37-

R4C4

0-R4

C46-

R4C4

7-R4

C48-

R4C4

9-R4

TNF-120572

BSA


12345670

10

20

30

40

50

60

70


Am

ino

acid

s pos

ition

Freq

uenc

y (

)







Clone Sequence T12














119889



119889TNF-120572 ratio of 002


50) A high IC

50


Clones

C2-R

3C1

8-R3

C22-

R3C2

5-R3

C30-

R3C3

3-R3

C41-

R3C4

2-R3

C45-

R3C4

6-R3

C32-

R4C3

3-R4

C35-

R4C4

6-R4

C47-

R4

1119864minus021119864minus011119864+001119864+011119864+021119864+031119864+041119864+051119864+061119864+07

119870119889

BSA

119870119889

TNF-120572


119889BSA119870

119889







119870clone119889













108738

7202

619512

7176

7261

16528

25984

0 1000 2000 3000 4000 5000 6000

C2-R3 (C-GSKTQAP-C)








Kcloned


119870clone119889




119870clone119889



0

10

20

30

40

50

60

70

Rela

xivi

ty(sminus1

m

Mminus1)














4 Discussion



(a) (b)

(c) (d)

(e) (f)











119870lowast









Acknowledgments


References







































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Clone Sequence T12














119889



119889TNF-120572 ratio of 002


50) A high IC

50


Clones

C2-R

3C1

8-R3

C22-

R3C2

5-R3

C30-

R3C3

3-R3

C41-

R3C4

2-R3

C45-

R3C4

6-R3

C32-

R4C3

3-R4

C35-

R4C4

6-R4

C47-

R4

1119864minus021119864minus011119864+001119864+011119864+021119864+031119864+041119864+051119864+061119864+07

119870119889

BSA

119870119889

TNF-120572


119889BSA119870

119889







119870clone119889













108738

7202

619512

7176

7261

16528

25984

0 1000 2000 3000 4000 5000 6000

C2-R3 (C-GSKTQAP-C)








Kcloned


119870clone119889




119870clone119889



0

10

20

30

40

50

60

70

Rela

xivi

ty(sminus1

m

Mminus1)














4 Discussion



(a) (b)

(c) (d)

(e) (f)











119870lowast









Acknowledgments


References







































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


108738

7202

619512

7176

7261

16528

25984

0 1000 2000 3000 4000 5000 6000

C2-R3 (C-GSKTQAP-C)








Kcloned


119870clone119889




119870clone119889



0

10

20

30

40

50

60

70

Rela

xivi

ty(sminus1

m

Mminus1)














4 Discussion



(a) (b)

(c) (d)

(e) (f)











119870lowast









Acknowledgments


References







































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b)

(c) (d)

(e) (f)











119870lowast









Acknowledgments


References







































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








119870lowast









Acknowledgments


References







































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article phage display screening for tumor necrosis...

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