3226 FIPFDPMSMRWE[0.0057,NT] SYIEPPERHRHR[0.0230,57] DDAKSRQGPLFR[0.0062,NT] NVNEGKAGVTGW[0.0036,NT] 4 Phage display (subtractive panning) 5 PMID:27989733 Myeloperoxidase Anti-human myeloperoxidase antibody Not determined. Not determined. Ph.D.-12 phage display library (X12) Surface plasmon resonance (SPR),Quartz crystal microbalance (QCM) A quartz crystal microbalance sensor (QCM D-300; Q-sense, Sweden) equipped with a QSX 301 gold chip was used to determine the molar binding ratio of hMPO to the immobilized ligand, i.e. the anti-hMPO antibody or the synthesized peptide. To study the binding kinetics and equilibrium, a surface plasmon resonance (SPR) biosensor (Biacore 3000, GE Healthcare, USA) equipped with a CM5 Sensor chip was used. The first colum of the affinity values represents the molar binding ratio determined by QCM, the second column denotes the Kd values (nM) determined by SPR. Before proceeding to the next round of biopanning, non-specific phages that were included in the selected phages were removed by a ‘subtraction’ (or negative selection) step by using epoxy-functionalized magnetic particles without a myeloperoxidase from human leukocytes (hMPO) coating (‘nude’ magnetic particles). The supernatant from this subtraction step was introduced to the next round. We selected and chemically synthesized a 12-mer peptide (SYIEPPERHRHR). Quartz crystal microbalance and surface plasmon resonance analyses revealed that the molar binding equilibrium ratio of the synthesized peptide was 0.023, approximately 43-fold lower than that of the anti-hMPO antibody. The dissociation constant (Kd) was 57 nM, which was comparable to that of the native antibody (83 nM). We biotinylated the peptide at its N-terminus and attached the biotinylated peptide to the surface of streptavidin-coated magnetic particles to assess its ability to selectively capture hMPO. The binding equilibrium data were similar to the previous analyses; specifically, around 0.021 mol peptide bound to 1 mol of hMPO. Antigen capture was found to be selective and to be relatively little influenced by the presence of human serum albumin (HSA), an abundant constituent of serum. 3227 HPNYDHDRMHTQ(16/50)[0.068] SMQYADHPVNTG(13/50)[0.272] FRVIYTDMPGAH(9/50)[0.250] 3 Phage display (common panning) 4 PMID:28346102 Pancreatic secretory granule membrane major glycoprotein GP2 Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) Saturation equilibrium assay To measure the binding strength of the selected peptide ligands to GP-2 (Kd, μM), saturation binding assays were performed. Binding affinity of each peptide was calculated by nonlinear regression and transformed to Scatchard plot using the GraphPad Prism 6 program (GraphPad Software, La Jolla, CA). Four rounds of biopanning were performed using a 96-well plate (Corning, Corning, NY). Selected peptides were conjugated to the C-terminal of enhanced green fluorescence protein (EGFP) and evaluated for their ability to induce an immune response in mice. One of our selected peptides, Gb-1 (HPNYDHDRMHTQ), showed high binding affinity to GP-2 and, when fused to EGFP, significantly increased the uptake of EGFP by M cells compared to EGFP alone. After oral administration, the Gb1-EGFP fusion induced efficient mucosal and systemic immune responses in mice measured at the level of antigen-specific serum and fecal antibodies, cytokine secretion, and lymphocyte proliferation. Furthermore, the IgG subclasses and cytokine secretion showed that ligand Gb-1 induced a Th2-type immune response. 3228 CAVECPSTLDKH[5.05, 4.35] LSSNPKLYVGLE[5.32, 4.42] 2 Phage display (subtractive panning) 2 PMID:28434129 Human IgG anti-HEV positive serum Capsid protein Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA The sandwich phage ELISA was used to test mimotope reactivity against anti-HEV positive sera S51 and S56, respectively. The ratio of clone OD absorbance and wt (wild-type phage M13KE) OD absorbance at 450 nm was calculated. The first column of the affinity values is the ratio with S51, the second column is the ratio with S56. The positive selection was carried out with protein G magnetic micro beads (Miltenyi Biotec) labelled with human IgG anti-hepatitis E virus (HEV) positive serum. The eluted bound phage was negatively selected with beads labelled with human IgG anti-HEV negative serum. Unbound phage from negative selection was amplified in E. coli ER2738 by a high-throughput method [38] and subjected to one more round of biopanning with different positive and negative sera. Four mimotopes did not have homology with the primary sequence of HEV ORF2 capsid but competed effectively with a commercial HEV antigen for binding to anti-HEV reference serum. When the reactivity profile of each mimotope was compared with Wantai HEV-IgG ELISA, the most sensitive HEV immunoassay, mimotopes showed 95.2–100% sensitivity while the specificity ranged from 81.5 to 95.8%. PepSurf algorithm was used to map affinity-selected peptides onto the ORF2 crystal structure of HEV genotype 3, which predicted that these four mimototopes are clustered in the P domain of ORF2 capsid, near conformational epitopes of anti-HEV neutralising monoclonal antibodies. These HEV mimotopes may have potential applications in the design of structural vaccines and the development of new diagnostic tests. 3229 HCSSAVGSWTWENGKWTWKGIIRLEQ[65] CSSPIQGSWTWENGKWTWKGIIRLEQ[16] HSCSSPIQGSWTWENGKWTWKGIIRLEQQP[3] 3 Phage display (common panning) 4 PMID:22271427 Fibronectin extradomain B, EDB Not determined. Not determined. Not determined. Aptide pIGT2-based phage display library (X6GSWTWENGKWTWKGX6) Surface plasmon resonance (SPR) Kinetic binding data (Kd, nM) for selected aptides, as determined by affinity measurements using BIAcore chips adapted for surface plasmon resonance. To immobilize the target protein (biotin-fibronectin extradomain B), streptavidin was immobilized directly on 96-well plates overnight at 4°C, and then the target protein was immobilized on the plate-bound streptavidin. 3230 HANFFQGSWTWENGKWTWKGWKYNQS[30] 1 Phage display (common panning) 4 PMID:22271427 Vascular endothelial growth factor A, VEGF-A Vascular endothelial growth factor receptor 1, VEGFR-1 1FLT,1QTY, Not determined. Aptide pIGT2-based phage display library (X6GSWTWENGKWTWKGX6) Surface plasmon resonance (SPR) Kinetic binding data (Kd, nM) for selected aptides, as determined by affinity measurements using BIAcore chips adapted for surface plasmon resonance. To immobilize the target protein, streptavidin was immobilized directly on 96-well plates overnight at 4°C, and then the target protein was immobilized on the plate-bound streptavidin. 3231 ASTINFGSWTWENGKWTWKGYTRRWN[93] 1 Phage display (common panning) 4 PMID:22271427 T-cell antigen CD7 Not determined. Not determined. Not determined. Aptide pIGT2-based phage display library (X6GSWTWENGKWTWKGX6) Surface plasmon resonance (SPR) Kinetic binding data (Kd, nM) for selected aptides, as determined by affinity measurements using BIAcore chips adapted for surface plasmon resonance. To immobilize the target protein, streptavidin was immobilized directly on 96-well plates overnight at 4°C, and then the target protein was immobilized on the plate-bound streptavidin. 3232 CSQNAYGSWTWENGKWTWKGWLYQNF[71] 1 Phage display (common panning) 4 PMID:22271427 His6-tag Not determined. Not determined. Not determined. Aptide pIGT2-based phage display library (X6GSWTWENGKWTWKGX6) Surface plasmon resonance (SPR) Kinetic binding data (Kd, nM) for selected aptides, as determined by affinity measurements using BIAcore chips adapted for surface plasmon resonance. To immobilize the target protein, streptavidin was immobilized directly on 96-well plates overnight at 4°C, and then the target protein was immobilized on the plate-bound streptavidin. 3233 PD(93.33%) DT(6.67%) 2 Phage display (common panning) 3 PMID:28548828 Gold nanocube Not determined. Not determined. Not determined. pVIII AE(X)2DP phage display library (AEX2DP) This clone displayed Pro–Asp on the surface of the bacteriophage and had amplification yields similar to those of the wild-type helper bacteriophage (VCSM13). The clone displayed even binding of gold nanocubes along its length and minimal aggregation after binding. We conclude that, for efficient amplification, the exposed pVIII amino acid length should be limited to six residues and Ala–Glu–Pro–Asp–Asp–Pro (AEPDDP) is the ideal fusion protein sequence for guaranteeing the optimal formation of a complex with gold nanocubes. 3234 SDG DID DVS ESQ ENS RMG DNA EVP 8 Phage display (common panning) 3 PMID:28548828 Gold nanocube Not determined. Not determined. Not determined. pVIII AE(X)3DP phage display library (AEX3DP) 3235 AGDS 1 Phage display (common panning) 3 PMID:28548828 Gold nanocube Not determined. Not determined. Not determined. pVIII AE(X)4DP phage display library (AEX4DP) 3236 FRGDKMQL(7) HWFWSKWW(5) WWKFMWFQ(1) RAWWSHWH(1) WRWASWFW(1) WHQNIHSP(1) 6 Phage display (common panning) 12 PMID:28468949 Head and neck squamous cell carcinoma (HNSCC) cell line HNO97 Not determined. Not determined. Not determined. SFTI-based phage display (SFTI8Ph) library (X8) Initially, phages were incubated for 1 hour with HNO97 grown to 90% confluence. Unbound phages were removed by washing steps and the cells were lysed with 1% Triton X-100 solution. Phages isolated from cell lysate were amplified and packaged in XL1 blue bacteria overnight and precipitated in polyethylenglycole solution . Subsequently, the phages were exposed alternatingly to HNO97 cells and HNO97 protein fractions (100nmol/L) in96-well plates for1 hour. After 5 PBS washing steps phages were eluted in 100 mLGlycin/HCl (pH 2.2) per well, neutralized by 15 mL Tris-HCl(pH 9.1) and amplified in XL1 blue bacteria. For titration, the phages were diluted and grown on agar plates. Twelve selection rounds were performed followed by single stranded DNA isolation of 16 clones (QIApreo Spin M13 Kit;Qiagen). 3237 DWYRDPVFDVLL LWRRVVVDSYGV YMWIACPDGWDC WWDWAVQSETLL WWVNPLGVTQDV EYPWWMEQFYLE WWAEAWFAWTPG ISWHSDWWVELV 8 Phage display (common panning) 4 PMID:28701344 The μ (mu) homology domain of suppressor of yeast profilin deletion Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) From the screen, we identified eight potential Syp1 ligand sequences and, to aid in the search for potential motifs, we compared the peptide sequences to that of Mid2 (251-316), the smallest fragment of Mid2 previously shown to bind the Syp1 μHD. Two DxY sequences (DWYRDPVFDVLL and LWRRVVVDSYGV), two YxxΦ motifs (YMWIACPDGWDC and EYPWWMEQFYLE), and several instances of two consecutive residues with bulky side chains, including one WY (DWYRDPVFDVLL) and five WW sequences (WWDWAVQSETLL, WWVNPLGVTQDV, EYPWWMEQFYLE, WWAEAWFAWTPG and ISWHSDWWVELV), were present in the peptides. All of these potential Syp1-interacting motifs are present in Mid2 (251-316). 3238 NPNSAQG 1 Phage display (common panning) 3 PMID:28710679 Cellular tumor antigen p53 Not determined. Not determined. Not determined. Ph.D.-7 phage display library (X7) ELISA ELISA of phage against p53 domains was carried out by coating 100 µl of 10 µg/ml of peptide solution (in 2% non-fat dry milk in 1x PBS) overnight at 4 °C. Reaction is read using a microplate reader set at 410 nm. The estimated gain of signal (% phage/control) for the phage N6 (with the sequence of NPNSAQG) is 48.23%. Three panning rounds were performed directly on the fusion GST-p53 that was previously fixed on G-Sepharose beads. Phages were sequenced and shown to contain a consensus sequence NPNSAQG 3239 QVNGMLGERSQQ(5)[0.77, 0.21] VYPYPKVDLSQD(3)[0.64, 0.28] DVSSLYDGPVGD(2)[0.64, 0.27] HAQGSLGTPVFM(2)[0.83, 0.24] Y-x(2)-P-x-G-D-L-G 4 Phage display (common panning) 3 PMID:28711695 Anti-FMDV monoclonal antibody 6C9 Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA Four phage clones were selected after three rounds of biopanning, and their binding activity to MAb 6C9 was analyzed in phage ELISA. Murine MAb 5D12 and wild-type M13 phage (PL) were used as negative controls. For each clone, the absorbance at 450 nm was calculated as an average of three duplicates. Data shown were absorbance values (the first column of the affinity values) reproduced from Figure 2. Additionally, binding inhibition of phage clones and whole virus antigens to MAb 6C9 were analyzed in competitive ELISA. TBS was used as a negative control. For each clone, the absorbance at 450 nm was calculated as the average of three duplicates. Data shown were absorbance values (the second column of the affinity values) reproduced from Figure 3. The M13 phage display library displaying 12-mer random peptides (Ph.D-12 Phage Display Peptide Library Kit, New England Biolabs) was used. Affinity selection of the phage clones from the random peptide library was conducted following the manufacturer's recommendations with minor modifications. Screening of a phage-displayed random 12-mer peptide library revealed that MAb 6C9 bound to phages displaying the consensus motif YxxPxGDLG. 3240 DMHADHAVYTPR(13) DMHAEHPGYRTA(1) NIHPDPPVYTPR(1) NMHADHPGYRTR(1) DMHADHPGYRTG(1) DMHAEHPGDRTG(1) DMLADHPGDRTR(1) DMHADHPGDRTR(1) 8 Phage display (competitive panning) 5 PMID:28715634 Tumor necrosis factor receptor superfamily member 12A Tumor necrosis factor ligand superfamily member 12 Not determined. Not determined. Ph.D.-12 phage display library (X12) Considering the possible non-specific binding that emerges in the interaction between the phage library and L-Fn14 CRD, competitive elution was conducted to avoid the elution of background-bound phage. The natural ligand of Fn14, TWEAK, was served as the eluent. After five rounds of panning, twenty plaques were randomly selected and the amplified DNA was sequenced, of which thirteen binding clones gave rise to the following sequence: DMHADHAVYTPR. 3241 LANNMLTLSPKMPGGGYW(3) SPTIFSTLSPKMPGGGYW(3) AEAFAFTLSPKMPGGGYW(1) ATTLTTTLSPKMPGGGYW(1) AGDLQETLSPKMPGGGYW(1) ASSEIHTLSPKMPGGGYW(1) ASLPLMTLSPKMPGGGYW(1) SQAAMLTLSPKMPGGGYW(1) MTTTNETLSPKMPGGGYW(1) MFPANATLSPKMPGGGYW(1) SPIVPDTLSPKMPGGGYW(1) SPLLSSTLSPKMPGGGYW(1) VPQGNXTLSPKMPGGGYW(1) VPQGATTLSPKMPGGGYW(1) LPYTIPTLSPKMPGGGYW(1) GPHTLATLSPKMPGGGYW(1) DLLHWTTLSPKMPGGGYW(1) GSAAPETLSPKMPGGGYW(1) LPLAPYTLSPKMPGGGYW(1) TQLAASTLSPKMPGGGYW(1) SNSQAYTLSPKMPGGGYW(1) HLATPTTLSPKMPGGGYW(1) HNPIPYTLSPKMPGGGYW(1) 23 Phage display (in vivo) 3 PMID:28658990 The middle ear Not determined. Not determined. Not determined. X6TLSPKMPGGGYW M13 bacteriophage library (X6TLSPKMPGGGYW) 50ul aliquots of each 18-mer phage library containing approximately 50 copies of each distinct 18-mer phage peptide were applied to the external surface of intact TMs of six rats. The phage samples were then pooled, amplified in E. coli, and the resulting library applied to the TMs of additional rats for two more rounds of library enrichment. After the third round, 30 individual phage plaques were selected at random and sequenced to identify the expressed peptide. After a 15-minute incubation on the TM, the external ear canal was repeatedly rinsed with PBS to remove all bacteriophage. The rats were then sacrificed and the ME bullas harvested intact. The exterior surface of the bulla was then extensively rinsed to remove contaminating phage. The bulla was then opened, exposing the ME contents. Fluid was collected from the ME, diluted in PBS and bacteriophage were titered using E. coli ER2738 (New England Biolabs, Cambridge, MA). The phage samples were then pooled, amplified in E. coli, and the resulting library applied to the TMs of additional rats for two more rounds of library enrichment. Transport of phage bearing peptide TMT-3(18)-1 was 8.9-fold higher than that of the TMT-3 12-mer. Phage expressing peptide TMT-3(18)-2 exhibited a 3-fold enhancement, while the TMT-3(18)-3 phage exhibited a significant decrease. 3242 KISVYRSADSTKTTHLTL(26) TEVTSRSADSTKTTHLTL(3) EGNLFPSADSTKTTHLTL(1) 3 Phage display (in vivo) 3 PMID:28658990 The middle ear Not determined. Not determined. Not determined. X6SADSTKTTHLTL M13 bacteriophage library (X6SADSTKTTHLTL) 50ul aliquots of each 18-mer phage library containing approximately 50 copies of each distinct 18-mer phage peptide were applied to the external surface of intact TMs of six rats. The phage samples were then pooled, amplified in E. coli, and the resulting library applied to the TMs of additional rats for two more rounds of library enrichment. After the third round, 30 individual phage plaques were selected at random and sequenced to identify the expressed peptide. After a 15-minute incubation on the TM, the external ear canal was repeatedly rinsed with PBS to remove all bacteriophage. The rats were then sacrificed and the ME bullas harvested intact. The exterior surface of the bulla was then extensively rinsed to remove contaminating phage. The bulla was then opened, exposing the ME contents. Fluid was collected from the ME, diluted in PBS and bacteriophage were titered using E. coli ER2738 (New England Biolabs, Cambridge, MA). The phage samples were then pooled, amplified in E. coli, and the resulting library applied to the TMs of additional rats for two more rounds of library enrichment. Transport of phage bearing peptide TMT-3(18)-1 was 8.9-fold higher than that of the TMT-3 12-mer. Phage expressing peptide TMT-3(18)-2 exhibited a 3-fold enhancement, while the TMT-3(18)-3 phage exhibited a significant decrease. 3243 FRLGLLKAFRRLF[210] RRDMCSMYGLC[NT] CDYFALGWRCWL[NT] 3 Phage display (competitive panning) 4 PMID:28676225 CTP synthase 1 Not determined. Not determined. Not determined. T7 phage display library Surface plasmon resonance (SPR) SPR experiments were performed on a BiacoreT100 instrument equipped with SA sensor chips (GE Healthcare). Binding affinities (Kd) were calculated from the equation Kd = koff/kon. We evaluated the binding activity of synthetic peptides by SPR analysis. CTpep-3 (FRLGLLKAFRRLF) showed CTPS1-binding in a peptide concentration dependent manner, and the Kd value was estimated as 210 nM. At the 3rd and 4th rounds, phages were subtracted using cytidine triphosphate synthase 1 (CTPS2) immobilized magnetic beads. 3244 SADSTKTTHLTL(10/22) SPPGKFLESLRS(6/22) DVGAGRWFSDNG(4/22) TLSPKMPGGGYW(2/22) SDDSRPIAQFAI(NA) 4 Phage display (in vivo) 3 PMID:26946957 The middle ear Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) 50 μl aliquots of the naïve phage library, each containing 1,000 copies of the 2.9× 109 different peptide sequences in the library, were placed on the outer surface of the TM (Tympanic membrane) of anesthetized and immobilized rats with active OM (Otitis media), then laid on their side for 1 hr. Te remaining phage solution was removed by wicking and the TMs were then extensively washed with PBS several times to remove any unbound phage particles from the external canal and reduce the possibility of ME (Middle ear) sample contamination. The animals were then sacrificed by decapitation under deep anesthesia to isolate the ME from the single side where phage was applied. The bullas of the rats were then dissected, isolated, and further rinsed in PBS prior to opening, as another precaution against contamination. The bulla then was opened and ME effusion was collected in a sterile tube for further analysis. The number of phage particles present in the ME fluid sample was assessed using an aliquot of supernatant in standard plaque assay. Phage in the remaining fluid were amplified by infecting bacteria via standard methods29 to generate a library of candidate TM-translocating phage, which was re-applied onto the TMs again using the protocol described above. The screen was successively repeated twice before DNA sequencing. There was a strong selection for a central positively charged residue, lysine or arginine (K/R), in all five peptides. 3245 TPMVERNYNAAD(5/30) ALWPPNLHAWVP(3/30) THPSTKVPGTPA(3/30) TFNPPPPQMPST(3/30) ILVGDESDGAKP(2/30) MSTPLGQYTGTK(2/30) HYPTTQLPHHKQ(2/30) VQNSTLSTKMDS(2/30) MVEKYATLFQSS(1/30) SHQNYHLTPQPP(1/30) APTTQRVGWDQS(1/30) AHSANNFDVKGI(1/30) MPLMSEPALEML(1/30) NYEFSISEQSHD(1/30) VNPSSLHSHNHS(1/30) ATNVVIMTQPRP(1/30) 16 Phage display (in vivo) 10 PMID:26946957 The middle ear Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) In the first stage, the commercial phage library was applied onto the TM (Tympanic membrane) of ears with OM, allowed to incubate for 1 hour (hr), and the external canal was extensively rinsed with PBS to remove unbound phage as described earlier. The TM was then harvested from the ME (Middle ear) bulla, washed with PBS, and homogenized. Phage present in the homogenate was amplified, and the process was repeated twice to generate a library enriched for phage with TM-binding activity. We then exposed TMs of infected rats to this preselected library, followed by elution with low pH buffer to remove all phage that had not internalized into the membrane. The TM was again harvested by dissection, homogenized, and phage libraries were amplified in 3 consecutive rounds. The resulting “internalizing phage” selected library was again applied to the TM surface of ears for 1 hr in rats with OM (Otitis media). Fluids were then recovered from the opposite ME cavity side, phage within the samples were amplified, and re-applied for three additional rounds. 30 phage particles from the final two rounds of in vivo selection were chosen at random and sequenced to identify the peptides displayed by the transported phage. Using this successive selection procedure (ten rounds total), additional populations of peptide-targeted phage with the ability to bind, internalize and finally transit the TM were identified. Two animals per condition were used. Pooled samples recovered from the final round of in vivo selection were sequenced. Sorting the sequences into groups using ClustalW2 for alignment identified 5 major phylogenetic groups. Group 1 contained two peptides that are characterized by a conserved NxSTLSTK core motif. Group 2 had the noteworthy PPxx core motif and a variable C-terminal region. These were different from group 3 which appeared to have a PT(S/T)E(R/K) central motif preceding a variable C-terminal region. The fourth category of sequences contained a polar negatively charged N-terminal region followed by the consensus aliphatic motif SISEQxR though the C-terminal region. The final sequence group contained an interesting HxxYxxD motif and a C-terminal QxP motif. These results indicate that the different grouped peptides could be binding a variety of targets due to the differences in the consensus motifs and the physical and chemical characteristics of the amino acid groups involved. Many of the residues contained are polar, either negatively or positively charged, which suggests that electrostatic interactions are at play. 3246 DSQFNKYSIATV(2/19)[0.60] VQCRLGPPWCAK(1/19)[1.32] SLLHTSMPSMIA(1/19)[0.57] KVEPGTGAHSWQ(1/19)[0.55] VADSNWINPRGS(1/19)[0.52] QGPHEHTKIHID(1/19)[0.41] 6 Phage display (common panning) 3 PMID:28780344 Cholera toxin subunit B, CTX-B Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA ELISA signal was measured at 405nm using a microplate spectrophotometer (Multiskan FC, Thermo Scientific, Waltham, MA, USA). Binding affinity values shown were regenerated from Figure 1A. Biopanning over recombinant CTX-B led to rapid screening of a unique peptide with an amino acid sequence of VQCRLGPPWCAK, and the phage-displayed peptides analyzed using ELISA, were found to show specific affinities towards CTX-B. To address the use of affinity peptides in development of the biosensor, sequences of newly selected peptides were modified and chemically synthesized to create a series of affinity peptides.Performance of the biosensor was studied using plasmonic-based optical techniques: localized surface plasmon resonance (LSPR) and surface-enhanced Raman scattering (SERS). The limit of detection (LOD) obtained by LSPR with 3σ-rule was 1.89ng/mL, while SERS had a LOD of 3.51pg/mL. In both cases, the sensitivity was much higher than the previously reported values, and our sensor system was specific towards actual CTX-B secreted from V. cholera, but not for CTX-AB5. 3247 GVKCTWSSIVDWVCVDM(11)[7.3 ± 0.2] GTRCDWSAAYGWLCYDY(4)[NT] RSVCVWTAVTGWDCRND(1)[ 4.4 ± 0.1] x(3)-C-x-W-x(5)-W-x-C-x(3) 3 Phage display (subtractive panning) 4-5 PMID:28758361 Chicken egg yolk immunoglobulin, IgY Not determined. Not determined. Not determined. T7 X3CX9-10CX3 phage display library (X3CX9-10CX3) Surface plasmon resonance (SPR) Surface plasmon resonance (SPR) analysis was performed on a BIAcore T200 (GE Healthcare, Little Chalfont, UK). Binding kinetic parameters (Kd, μM) were calculated using BIAevalution Version3.2 Software (GE HealthCare). T7 phage libraries of X3CX7–10CX3 were incubated in wells coated with BSA to remove non-specific phage and then added to IgY-coated wells. The synthetic peptides (Y4-4: GVKCTWSSIVDWVCVDM, Y5-55: RSVCVWTAVTGWDCRND) showed high binding specificity to IgY-Fc and moderate affinity for IgY-Fc (Kd: Y4-4 = 7.3 ± 0.2 μM and Y5-55 = 4.4 ± 0.1 μM) by surface plasmon resonance analysis. To evaluate the ability to purify IgY, we performed immunoprecipitation and affinity high-performance liquid chromatography using IgYbinding peptides; the result indicated that these peptides can be used as affinity ligands for IgY purification. We then used Y4-4 peptide-conjugated column to purify IgY from egg yolks pre-treated using an optimized delipidation technique. 3248 CPFMSVANC(2) CAISSPGSC(1) CNAKHHPRC(1) CDSRWHSVC(1) CNGGYAFEC(1) CEHHKNTLC(1) CNSRLNQHC(1) CEIWSRAAC(1) CPGEPPTLC(1) CELQGWHLC(1) CPMFLASLC(1) CEPSLIPFC(1) CPRVNMLHC(1) CGENIQQYC(1) CQNHQSRHC(1) CGLNKTPGC(1) CRSDSYNKC(1) CGPKTPHKC(1) CSGALTSPC(1) CGSKLDALC(1) CSKQSTIAC(1) CHQWNSNSC(1) CSMEELVTC(1) CHTAHKATC(1) CSTEHTRSC(1) CKDQGQMRC(1) CTASPSGLC(1) CKPHTGHVC(1) CTDPNSTTC(1) CKPPGSYFC(1) CTNANSHLC(1) CLDSPNNVC(1) CTPDASRKC(1) CLKLTAASC(1) CTSNTKFNC(1) CLNDSVHQC(1) CTTYNATSC(1) CLNVPTKS(1) CWPPSTYSC(1) CLSDYSIMC(1) CYGKNNQRC(1) CMLNTSVAC(1) CYSELTGNC(1) 43 Phage display (common panning) 3 PMID:28757359 The human colon carcinoma Caco-2 cell Not determined. Not determined. Not determined. Ph.D.-C7C phage display library (CX7C) Phage displaying cyclic heptapeptide DNPGNET (DNP-phage) showed the greatest permeability across a Caco-2 cell monolayer and mouse intestinal epithelium. Interestingly, DNPGNET (DNP) does not contain any basic amino acids. Its isoelectric point (pI) was estimated to be 2.72. It did not reduce the viability or tight-junction integrity of Caco-2 cells at concentrations up to 100 μM for 24h. Up take of either DNP phage or fluorescence-labeled DNP derivative (AC-DNPGNET-CGGGS modified with 5/6-FAM at the C-terminal; the cysteines serve to generate the cyclic peptide via disulfide bond formation, and GGGS is the phage linker) by Caco-2 cells was inhibited by low temperature, unlabeled AC-DNPGNET-CGGGS and EIPA, a macropinocytosis inhibitor. Thus, DNP appears to facilitate transcellular permeability of phages via macropinocytosis, but not paracellular diffusion. These findings indicate that DNP is a promising candidate as a carrier to promote intestinal absorption of macromolecular drugs. 3249 KVVKTHR[1.34] MVYLTEK[0.49] KTVLTHR[0.28] NAGHLSQ[0.20] NMHTPMV[0.18] 5 Phage display (subtractive panning) 4 PMID:28744632 Tumor necrosis factor ligand superfamily member 10 Not determined. Not determined. Ph.D.-C7C phage display library (CX7C) ELISA Absorbance at 450 nm was measured on a Tecan microplate spectrophotometer (Genios). Data shown were regenerated from Figure 2b. For phage selection, phage particles were pre-incubated with mouse Fc. Then, bound phages were removed using magnetic beads with immobilized protein G (Invitrogen), while the supernatant was used for further selection of more specific peptides by adding mouse DR5-Fc. The cyclopeptide sequences selected from the library show appreciable homology at positions 2 (valine), 4 (leucine), and 5 (tyrosine). Substitution of valine for threonine in position 2 in a KVVLTHR peptide leads to a decrease in binding. It is possible that these very amino acids play the key role in interaction between the peptide and DR5. 3250 CLPTKFRSC(5)[high binding, 270 ± 151] CHEQQNPHC(1)[low binding, NT] CHKHKDSQC(1)[low binding, NT] CHLGEMGHC(1)[low binding, NT] 4 Phage display (common panning) 3 PMID:28733706 The extracellular domain of receptor tyrosine-protein kinase erbB-3 Not determined. Not determined. Not determined. Ph.D.-C7C phage display library (CX7C) ELISA Absorbance at 405 nm was read by the spectrophotometer. Peptide ELISA with HER3P1 (CLPTKFRSC) demonstrates an affinity of 270 ± 0.151 nM. The receptor tyrosine-protein kinase erbB-3 extracellular domain (R&D Systems, Minneapolis, MN) was conjugated with long chain biotin (Thermo Scientific, Waltham, MA) utilizing standard NHS ester chemistry and purified. Purified, biotinylated receptor tyrosine-protein kinase erbB-3 was bound to Dynabeads M-280 streptavidin beads (Thermo Scientific). The selection converged to a consensus peptide sequence that was subsequently found to bind receptor tyrosine-protein kinase erbB-3 (HER3) with an affinity of 270 ± 151 nM. The peptide with the sequence of CLPTKFRSC, termed HER3P1, was bound with high selectivity to HER3 over other similar receptor tyrosine kinases such as EGFR and HER2. Furthermore, HER3P1 was able to distinguish between high and low HER3-expressing cells in vitro. The peptide was radiolabeled with Ga-68 and demonstrated to specifically bind HER3 by in vivo PET imaging. Uptake of [ 68Ga]HER3P1 was highly specific for HER3-positive tumors, with tumor-to-background ratios ranging from 1.59–3.32, compared to those of HER3-negative tumors, ranging from 0.84–0.93. The uptake of [68Ga]HER3P1 also demonstrated high (P < 0.001) correlation with protein expression as quantified by Western blot and confirmed by biodistribution. HER3P1 accurately quantifies expression of HER3 by PET imaging and has potential utility as a clinical imaging agent.