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A novel peptide defined through phage display for therapeutic protein and vector neuronal targeting

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A novel peptide defined through phage display for therapeutic protein and vector neuronal targeting
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  A novel peptide defined through phage display for therapeutic proteinand vector neuronal targeting James K. Liu, a  Qingshan Teng, a  Mary Garrity-Moses, a  Thais Federici, a  Diana Tanase, a  Michael J. Imperiale,  b and Nicholas M. Boulis a , T a   Department of Neuroscience and Center for Neurological Restoration, NB2-126, Cleveland Clinic Foundation, 9500 Euclid Avenue,Cleveland, OH 44195, USA  b  Department of Microbiology and Immunology, University of Michigan School of Medicine, Ann Arbor, MI 48109, USA Received 25 August 2004; revised 1 December 2004; accepted 20 January 2005Available online 18 March 2005 A novel peptide with the binding characteristics of tetanus toxin wasidentified with phage display, for application in therapeutic protein andvector motor and sensory neuron targeting. A 12mer phage library wasbiopanned on trisialoganglioside (G T1b ) and eluted with the tetanustoxin C fragment (rTTC). Phage ELISAs revealed increases in G T1b binding for the Tet1 and Tet2 phage clones when compared topeptideless phage (PLP). rTTC displaced both Tet1 and Tet2 phageclones from G T1b , and both clones reduced rTTC-G T1b  binding.Comparison of Tet1, Tet2, PLP, and the random phage library bindingto PC12 and HEK293 cells revealed enhanced cellular binding by Tet1and Tet2 phage. Tet1 phage binding was selective for neurons.Immunofluorescence also confirmed selective PC12 binding of Tet1and Tet2 phage. Fluorescein-conjugated synthetic Tet1, but not Tet2,peptide showed strong binding to cultured PC12, primary motorneurons, and dorsal root ganglion (DRG) cells. Synthetic Tet1 boundDRG and motor neurons but not muscle in tissue sections. Theenhanced neuronal binding affinity and specificity of Tet1, a novel 12amino acid peptide, suggests potential utility for targeting neuro-therapeutic proteins and viral vectors in the treatment of motor neurondisease, neuropathy, and pain. D  2005 Elsevier Inc. All rights reserved.  Keywords:  Dorsal root ganglion; Ganglioside; Motor neuron; Phagedisplay; Tetanus toxin C fragment  Introduction Currently, treatment for Amyotrophic Lateral Sclerosis (ALS) islargely palliative (Borasio and Miller, 2001). The administration of  neurotrophic growth factors as a means to protect motor neuronshas been extensively explored (Apfel, 2001). Ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), insulin-like growth factor (IGF), and glial cell-line-derived neurotrophicfactor (GDNF) slow motor neuron degeneration (Askanas, 1995;Wang et al., 2002). Insulin-like Growth Factor-I (IGF-I) protectsmotor neurons (MNs) in both organotypic slice as well asdissociated cultures (Bilak and Kuncl, 2001; Vincent et al.,2004). Based on these results, clinical trials of subcutaneous andintrathecal neurotrophic factors were attempted in ALS. However,CNTF peripheral injections caused weight loss, fever, cough, andfatigue (CNTF, 1996; Miller et al., 1996) and subcutaneous injections of BDNF caused injection site reactions, changes in bowel function, and other behavioral side effects (BDNF, 1999;Bradley and Group, 1995). Similarly, recombinant GDNF pre-sented a short plasma life, poor access to motor neurons, andsevere side effect (Alisky and Davison, 2000; Haase et al., 1997).Trials of IGF-I in sporadic ALS have had mixed results (Borasio et al., 1998; Lai et al., 1997). The failure of these many trials may beattributed in part to insufficient trophic factor delivery to motor neurons and nonspecific delivery to non-motor systems.Viral gene transfer may offer an alternative approach to motor neuron protection with several advantages over protein-basedtherapies. Gene-based therapies allow for prolonged expression of neuroprotective factors minimizing or potentially eliminating theneed for repeated dosing. Several viral vectors have beendemonstrated to undergo retrograde axonal transport, lending themto application in motor neuron therapies (Glorioso et al., 1995;Mazarakis et al., 2001). Our laboratory (Boulis et al., 2003a,b) and others (Kaspar et al., 2002) have recently characterized the retrograde axonal transport of adeno-associated virus (AAV)vectors from peripheral injection sites to spinal motor neurons.Dramatic therapeutic effects have recently been achieved byemploying this approach to IGF-I gene delivery in the SOD1mouse model of ALS (Kaspar et al., 2003). However, the authors  point out that a small fraction of peripherally administered vector reaches spinal motor neurons, and some investigators have failed to 0969-9961/$ - see front matter   D  2005 Elsevier Inc. All rights reserved.doi:10.1016/j.nbd.2005.01.022  Abbreviations:  G T1b , trisialoganglioside; AAV, adeno-associated virus;rTTC, recombinant tetanus toxin C fragment; PLP, peptideless phage; RL,random library; PC12, pheochromocytoma cells; DRG, dorsal root ganglion; MN, motor neuron. T  Corresponding author. Fax: +1 216 445 1466.  E-mail address:  boulisn@ccf.org (N.M. Boulis). Available online on ScienceDirect (www.sciencedirect.com). www.elsevier.com/locate/ynbdi Neurobiology of Disease 19 (2005) 407–418  detect this phenomenon (Chamberlin et al., 1998; Martinov et al.,2002). This poor efficiency may result in part from a tendency of various AAV serotypes to bind and infect the muscle cells that surround the axon terminal at the neuromuscular junction (Abadieet al., 2002; Rohr et al., 2002). Alternatively, low axon terminal binding affinity could undermine effective motor neuron genedelivery. Thus, like trophic factor therapies, AAV therapies may belimited by inefficient binding to motor neuron terminals andnonspecific binding to surrounding cell types.Because AAV is capable of infecting a large range of host cells, poor specificity presents a challenge to application in a variety of organ systems. To circumvent this disadvantage, the capsid of AAV has been modified to allow for targeted vector tropism (Girodet al., 1999). Models of the AAV capsid proteins provided by X-raycrystallography have defined loop and terminus regions of the protein which appear on the vector’s surface (Xie et al., 2002). The insertion of peptides with specific binding affinit ies into theseregions of the capsid can alter the vector’s tropism (Grifman et al.,2001; Loiler et al., 2003; Nicklin et al., 2000; Shi and Bartlett,2003; Wu et al., 2000). Warrington et al. (2004) point out that  successful insertion into the loop regions of AAV responsible for heparin sulfate proteoglycan requires the use of peptides that areless than 30 amino acids in length. Indeed, almost all successfulretargeting through capsid mutation has involved the use of small peptides. Larger peptides may be inserted into the residue 138 of the VP1 and VP2 capsid proteins (Warrington et al., 2004), but this technique requires a capsid complementation that may pose achallenge for manufacturing large-scale preps for clinical applica-tion. The challenge of enhancing motor neuron binding of therapeutic proteins may be similarly constrained. That is, fusionof trophic factors to larger proteins could alter their folding andconformation, undermining their neuroprotective properties. Theseconcerns have motivated our search for small peptides withenhanced neurotropism.Phage display is an effective method for isolating novel peptides with specific binding properties. In this technique, aconstrained or random library of oligonucleotides is inserted intoone of the genes encoding phage coat proteins. The resultinglibrary of phage presents the peptides encoded by the oligonucleo-tides on their surface, creating a physical link between the DNAsequence and the binding properties of the encoded peptide.Biopanning strategies that select for specific binding properties arerepeated to enrich for phage presenting peptides with these properties (Fig. 1A). Phage display has been used to isolate peptides with the capacity to bind efficiently and specif ically toseveral cell types, including fibroblasts (Barry et  al., 1996), muscle cells (Samoylova and Smith, 1999), microglia (Samoylova et al., 2002), lung tumor  cells (Oyama et al., 2003), neuroblastoma (Zhang et al., 2001), and vascular endothelial cells ( Nicklin et al., 2000). Peptides with specific binding to endothelial cells defined by phage display have been used to create AAV with enhancedendothelial gene delivery ( Nicklin et al., 2001; White et al., 2003). We have recently reported the use of phage display to identify 12amino acid peptides wit h affinity for neuronal membrane ganglio- sides (Liu et al., 2004). Hou et al. (2004) have also recently identified a group of 12 amino acid peptides with cerebellar granule cell binding affinity.To define peptides with affinity for motor neuron axonterminals, a biopanning strategy was developed to select for the binding characteristics of tetanus toxin. The botulinum and tetanusclostridial toxins are the most potent neurotoxins identified(Middlebrook, 1989). Their remarkable toxicity results from thecombination of their neurospecificity, neuronal binding affinity,and the extraordinar y potency of their intracellular activity (Ka;10  12 to 10  13 M) (Middlebrook, 1989; Montecucco et al., 1991).High-affinity selective neural uptake of CNTs derives from the Cfragment of the toxins’ heavy-chain component. The toxinsselectively bind and penetrate axon terminals in the peripheralnervous system. Tetanus toxin penetrates the terminals of all peripheral neurons including autonomic, sensory, and MNs(Stoeckel et al., 1975). Uptake of the labeled C fragment of  tetanus toxin occurs most avidly in large motorneurons, followed by preganglionic neurons, with t he least uptake in sensory neurons(Fishman and Carrigan, 1988). Tetanus neuronal binding and  biological activity depends on the trisialoganglioside (G T1b )receptor (Williamson et al., 1999). In order to select for phage with tetanus-like binding properties,a type III 12mer phage library presenting 10 9 random 12 amino acid peptides on the pIII phage coat protein was exposed to G T1b -coated plates and eluted with glycine-hydrochloric acid. Recovered phagewere amplified and underwent three more rounds of biopanning,utilizing recombinant tetanus C fragment (rTTC) to elute phage.The oligonucleotide inserts of forty-two phage plaques from theresulting library were excised and sequenced. Four sequences werefound to occur more than once, and a single sequence (Tet1) wasfound in 83% of plaques sequenced (Fig. 1A). The Tet1 phageclone and a second clone with limited homology (Tet2) wereevaluated by anti-phage ELISA for G T1b  binding and background plate binding. While both clones possessed enhanced G T1b  binding,Tet1’s selectivity for G T1b  was greater than Tet2. Next, theindividual clones were assessed for competition with rTTC for G T1b  binding. rTTC competed with both clones for binding to G T1b . Next, cellular binding of the phage peptides were compared to binding by the random library and peptideless phage (PLP). Boththe recovery of infectious phage and immunofluorescence suggest selective binding to differentiated pheochromocytoma cells (PC12)in comparison with kidney epithelial cells (HEK293). As with G T1b  binding, Tet1 selectively bound the neuronal cell line with greater efficiency and specificity than Tet2. Synthetic Tet1 and Tet2fluorescein-conjugated peptides were constructed and allowed to bind to several different neuronal cells lines. Tet1, but not Tet2, bound avidly to PC12 cells, primary motor neurons, and dorsal root ganglion (DRG) cells. These findings suggest that Tet1 may provide a means to achieve enhanced and selective neural bindingof both viral vectors and neuroprotective proteins. Materials and methods  Phage display biopanning against immobilized trisialogangliosides (G  T1b  ) Trisialoganglioside (Sigma-Aldrich Co., St. Louis, MO) dilutedin 0.1 M NaHCO 3  pH 8.6 were immobilized onto 96-well plates byincubating wells with 100  A g/ml overnight at 4 8 C. The wells werethen blocked overnight with phage block buffer (0.1 M NaHCO 3  pH8.6, 5 mg/ml BSA, 0.02% NaN 3 ) at 4 8 C. The blocking buffer was discarded and the wells were washed 6 times in TBS with0.1% Tween-20. Each well was exposed to 4    10 10  plaqueforming units (pfu) of a random 12 amino acid (12mer) pIII peptide phage library and allowed to incubate for 1 h. After washing awayunbound phage, bound phage were eluted with a nonspecific  J.K. Liu et al. / Neurobiology of Disease 19 (2005) 407–418 408  general elution buffer, containing 0.2 M Glycine–HCl (pH2.2) and1 mg/ml BSA for 1 h, following neutralization with 1 M Tris–HCl(pH 9.1). The nonspecific eluates from three separate wells werecombined and amplified in ER2738  E. coli  cells and titered. For the second through fourth rounds of biopanning, G T1b  wasimmobilized and blocked as stated above. Following incubationwith 10 11  pfus of the previous rounds’ eluate, bound phage waseluted with 100  A g/ml of rTTC (Roche Diagnostics Corp., Fig. 1. Phage display biopanning strategy. (A) Schematic depicting a single round of biopanning. (B) Flow chart detailing the four-staged biopanning. Theinitial round utilized pooled phage eluted from three wells with Glycine–HCl (pH 2.2), and three subsequent rounds eluted with rTTC.  J.K. Liu et al. / Neurobiology of Disease 19 (2005) 407–418  409  Indianapolis, IN) for 2 h. Following the fourth round of  biopanning, the enriched phage pool was plated onto LB/IPTG/ Xgal plates and 50 colonies were picked for plasmid preparation.Automated sequencing was performed by SeqWright, Inc. (Hous-ton, TX). The phage display biopanning process is schematicallyillustrated in Fig. 1. G  T1b  binding, competition, and displacement ELISAs The dominant phage clone identified in 83% of sequencedcolonies (Tet1) and a second clone bearing homology with Tet1(Tet2) were amplified for binding studies. G T1b  was immobilizedand blocked as mentioned previously. For binding studies, 10 9  pfuof individual phage clones were applied to ELISA wells coatedwith G T1b . Phage were allowed to bind for 2 h. The wells werewashed in TBST (TBS + 0.5% Tween 20) and then incubated withmouse anti-M13 HRP-conjugated antibody (Amersham PharmaciaBiotech Inc., Piscataway, NJ). Secondary antibody was developedwith OPD substrate (Pierce, Rockford, IL) and plates were readusing a SpectraMax 190 microplate reader at an absorbance of 490 nm. The signal of bound phage in G T1b -coated wells wasdivided over signal from uncoated wells to obtain the percentageincrease in binding due to the presence of G T1b . In addition, Tet1and Tet2 binding in G T1b -coated wells were compared to peptideless phage (PLP) to obtain the increase in bindingattributable to selected peptides. Next, we performed phage clone-rTTC competition studies.Individual phage clones were applied at a titer of 10 9  pfu G T1b -coated ELISA wells with varying concentrations of rTTC (0, 25,50, and 75 ng/ml). Phage and rTTC were incubated for 2 h. Phage binding was measured using mouse anti-M13 HRP-conjugatedantibody, developed with OPD substrate and measured as previously described. In addition, the inverse experiment was performed, displacing rTTC from G T1b  with individual phageclones. For rTTC displacement, rTTC was allowed to bind for 2 hto G T1b -coated wells. Following removal of rTTC, the wells wereincubated with phage clones for 2 h at 10 9  pfu. Anti-tetanus toxinC fragment (Roche, Indianapolis, IL) was applied for 1 h followed by a 1-h incubation with goat anti-mouse IgG 1 -FITC secondaryantibody (Santa Cruz Biotechnology, Inc, Santa Cruz, CA).Secondary antibody was developed with OPD substrate andmeasured as previously described. The percentage decrease inrTTC signal from non-phage exposed wells to phage-displacedrTTC was calculated. Cell binding assays Protocols for cell binding assays were adapted from Samoylovaet al. (2002). PC12 cells and HEK293 were grown on collagen-coated plates in DMEM supplemented with 10% horse serum and5% FBS. PC12 cells were differentiated with 100 ng/ml NGF 2.5S(Invitrogen Corp., Carlsbad, CA) in DMEM with 2% horse serum,and 1% FBS. Binding assays were performed 3 days after differentiation. 10 9  pfu of individual phage clones were appliedto the cells in phage wash buffer (PBS, 0.1% BSA, 1 mM CaCl 2 ,10 mM MgCl 2 ) for 1 h. Wells were exposed to Tet1, Tet2, PLP phage clones, or the random 12mer phage library used to initiate biopanning. The cells were then washed 6 times, for 5 min per wash, with phage wash buffer. The cells were then lysed in lysis buffer (1% Triton 100, 10 mM Tris, 2 mM EDTA) for 1 h at roomtemperature. The lysis buffer was collected and the bound phagewas titered. The same protocol was repeated for HEK293 cells.The  b  bound phage ratio  Q   is defined as the ratio of phage retrievedfollowing cell lysis divided by the amount of phage applied to thecells (output/input ratio). Statistical analysis G T1b  binding studies and competition assays were replicated intriplicate and individual conditions were compared with a two-wayANOVA. Cell binding assays were performed on six wells of PC12cells and four wells of HEK 293 cells. Bound phage ratios for Tet1(  N   = 10), Tet2 (  N   = 10), peptideless phage (  N   = 10), and randomlibrary (  N   = 10) were compared in a two-way ANOVA evaluatingcell type and phage clone as separate variables. Student’s  t   testswere used to compare the binding of Tet1 and Tet2 clones to PC12and HEK293 cells.  Immunofluorescence phage localization in vitro The phage localization protocol was adapted from Samoylovaet al. (2002). HEK293 cells were grown as previously described.PC12 cells were grown on collagen-coated glass cover slips anddifferentiated as previously described. 10 9  pfus of individual phageclones were applied after fresh media was added to the cells andallowed to incubate overnight. HEK293 cells were exposed to Tet1and Tet2 phage, as well as PBS as a negative control. DifferentiatedPC12 cells were exposed to PBS, PLP, Random Library, Tet1, andTet2 phage. Following removal of media and PBS wash, cells werefixed in 4% paraformaldehyde. The cells were incubated for 15 minin wash buffer (PBS + 0.1% saponin). Cells were then blocked inPBS + 0.1% saponin + 1% BSA for 1 h at 4 8 C. Mouse anti-M13antibody (Amersham Pharmacia Biotech Inc., Piscataway, NJ)diluted 1:5000 in blocking buffer was applied to the cells for 1 h.Cells were rinsed three times with wash buffer for 5 min per wash.Goat anti-mouse I g G 2A  FITC-conjugated antibody (Santa CruzBiotechnology Inc, Santa Cruz, CA) was diluted 1:200 in blocking buffer and applied to the cells for 1 h. Cells were washed again andmounted with Vectashield mounting medium (Vector Laboratories,Inc., Burlingame, CA). Because Vectashield mounting mediumcontains DAPI stain, nuclear blue staining can be detected. Cellswere visualized using a Leica DM R microscope and pictures werecaptured with a QImaging Retiga EX camera.  Primary motor neuron and dorsal root ganglion culture Primary motor neuron culturing protocol was adapted fromVincent et al. (2004). Experiments were approved by the ClevelandClinic Foundation Animal Care and Use Committee and strictlyadhered to the requirements set forth by the  Guide for the Care and Use of Laboratory Animals  (National Academy of Sciences Press,Washington, DC, 1996). Spinal cords were obtained from 15-daySprague–Dawley rat embryos (Harlan, Indianapolis, IN). Dorsalroot ganglia and perineural membranes were removed and cordswere cut into approximately 2-mm sections. Cord sections were placed in trypsin/EDTA 1   solution. Cells obtained from thetrypsinized tissue were collected and centrifuged through a 6.8%metrizamide column for 15 min at 2000 rpm. The upper layer of the gradient was collected and diluted in Leibowitz L-15 mediafollowed by centrifugation. Pelleted cells were resuspended incomplete growth medium made in Neurobasal Medium with thefollowing supplements: 0.2  A g/ml  d -glucose, 2.5 mg/ml albumin,  J.K. Liu et al. / Neurobiology of Disease 19 (2005) 407–418 410  2.5  A g/ml catalase, 0.1 mg/ml biotin, 2.5  A g/ml SOD, 0.01 mg/mltransferrin, 5  A g/ml galactose, 6.3 ng/ml progesterone, 16  A g/ml putrescine, 4 ng/ml selenium, 3 ng/ml  h -estradiol, 4 ng/mlhydrocortisone, 2  A M  l -glutamine, penicillin/streptomycin/neo-mycin, and B-27. Cells were plated on glass cover slips in multi-well tissue culture plates pre-coated with poly- l -lysine. Mediumwas renewed after 1 h and neurite outgrowth was observed.DRGs collected from rat embryos were incubated in trypsin for 10 min at 37 8 C. Following addition of prewarmed calf serum tostop the trypsinization reaction, the cells were collected followingcentrifugation and resuspended in L-15 media. Following repeatedtrituration, cells were once again collected following centrifugationand resuspended in plating media (NB media supplemented with30 nm selenium, 10 nm hydrocortisone, 10 nm  h -estradiol, 10 mg/lapo-transferrin, pen/strep/neo, 12.5 ng/ml NGF, 0.05 mM FUDR,140  A M  l -glutamine). After incubation for 2 h to allow for cellattachment, the cells are incubated in feed media (plating mediawithout   l -glutamine) for culturing.  In vitro peptide binding  In vitro peptide binding protocol was adapted from Samoylovaet al. (2002). PC12, primary motor neurons, or DRGs werecultured as stated above. Following a 1-h incubation in serum-freemedia at 37 8 C, the cells were fixed in 4% paraformaldehyde for 20 min at room temperature. The cells were washed three times for 1 min each in PBS and then blocked in PBS with 0.1% BSA for 1 hat 4 8 C. Following an additional round of washing, fluorescein-conjugated Tet1 or Tet2 peptides (100  A g/ml), synthesized byEvoQuest Laboratory Services (Invitrogen Corp., Carlsbad, CA),were applied and allowed to bind cells for 1 h. Unbound peptideswere washed away and cells were mounted with Vectashieldmounting medium. Peptide binding was observed under fluores-cence as noted above.In a separate experiment, PC12 cells were grown to 80%confluency. Half of the wells were then exposed to NGF as previously described to induce differentiation. 24 h later, whenmorphological changes consistent with differentiation wereobserved in the NGF group, cultures were fixed and exposed toflourescein-conjugated Tet1 peptide as described. Cellular peptide binding in tissue sections Experiments were approved by the Cleveland Clinic Founda-tion Animal Care and Use Committee and strictly adhered to therequirement set forth by the  Guide for the Care and Use of   Laboratory Animals  (National Academy of Sciences Press,Washington, DC, 1996). Animals were anesthetized and underwent intracardiac perfusion with normal saline solution followed by buffered 2% paraformaldehyde. Brains and spinal cords wereremoved, 24 h postfixed in 2% paraformaldehyde, and cryopre-served in 20% sucrose for additional 24 h. Rodent DRG, lumbar spinal cord, and gastrocnemius muscle tissue sections frozen inoptimal cutting temperature gel (OCT; Sakura Finetek USA Inc,Torrance, CA) were cut at 20  A m in transverse sections on acryostat (Leica Microsystems, Nussloch, Germany). Sections werewashed in PBS for 5 min 3 times and placed on glass slides.Peptides (100  A g/ml) were added and allowed to remain for 1 h at room temperature. Specimens were washed for 5 min in PBS thencover slipped and mounted with Biomeda gel mount (Foster CityCa.) for microscopic fluorescent analysis. Results  Identification of G  T1b -specific peptides Peptides specific for G T1b  were isolated using a four-round biopanning strategy. The initial round of biopanning was carriedout in triplicate, eluting bound phage with a nonspecific acidicelution. This stringent elution was performed to prevent the lossof strong-binding phage clones in early rounds. Also, tooptimize the capture of G T1b  binding phage clones, eluatesfrom the three separate biopanned wells were combined andamplified for use in the second round of biopanning. In thesecond through fourth rounds of panning, the bound phage waseluted with rTTC to specifically isolate phage peptides that mimic tetanus toxin binding properties. Following the final biopanning round, 42 random phage clones were collected for sequence analysis of the peptide insert. A total of four different  peptide sequences was obtained (Table 1). 83% of these phage  plaques contained the same phage clone (Tet1). A secondsequence (Tet2) present in 5% showed a three amino acidcombination homology with Tet2. Both Tet1 and Tet2 contain aleucine, serine, and threonine combination, as well as sharingother amino acids such as histidine, tryptophan, and arginine. A blast search run on the sequences of Tet1 and Tet2 revealedhomology to a variety of oligonucleotides from unidentified proteins. Tet1 bore homology to an actin-reacting protein, not  previously associated with the nervous system. No similaritieswere detected between the selected peptides and the clostridialtoxins. G  T1b  binding and rTTC competition assays In order to confirm that phage peptides were selected due totheir affinity for G T1b  rather than from nonspecific plate binding,individual phage clones were applied to wells with and without G T1b  coating. Phage binding was measured via ELISA performed with an anti-M13 phage antibody. Fig. 2 demonstratesthat both Tet1 phage and Tet2 phage bound GT1b-coated plates better than uncoated plates, in contrast to PLP for which nosignificant difference in binding could be detected. Tet1 phage bound both uncoated and coated plates better than Tet2 phage.However, the difference in binding was greater for Tet1 phage.ANOVA revealed significant plate coating (  P   b  0.001), phageclone (  P   b  0.001), and coating by clone interaction (  P   b Table 1Peptide isolated through novel four-round biopanning processSequencing from 42 randomly selected clones revealed 4 different peptidesequences labeled Tet1–Tet4 identified in more than a single plaque. Aminoacid similarities between Tet1 and Tet2 are shown in bold in the left column. The right column contains the percentage of plaques in which theoligonucleotide sequence was detected.  J.K. Liu et al. / Neurobiology of Disease 19 (2005) 407–418  411
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