3426 GWVSNTTQAHHV(9) AMDIAYRTHREP(3) DLNKPKPLYQQH(2) ESMTYLSTAPEK(2) HNYPVLRPNQIT(2) APNAPTQTTTPV(1) ARHPDTNYSYGA(1) MMKQTDQLLRNN(1) NWHVYSTISNQT(1) TRTPPESYASVR(1) YSGKDLPPMKDS(1) 11 Phage display (competitive panning) 3 PMID:30705359 Chondroitin-4-sulfate, C4S Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) In each round of panning, binding phages were eluted with 100 μl of C4S (100 mg/ml in PBS) and applied to the next panning round. From the phage clones binding to C4S we selected three peptides (AMDIAYRTHREP, GWVSNTTQAHHV and TRTPPESYASVR) for further analysis. We observed that these peptides bind to C4S, but not chondroitin-6-sulfate, heparin sulfate or dermatan sulfate, in a concentration-dependent and saturable manner, whereas the scrambled peptides showed highly reduced or no binding to C4S. The C4S-binding peptides, but not their scrambled counterparts, when added to cultures of mouse cerebellar neurons and human neuroblastoma cells, neutralized the inhibitory functions of the C4S- and CSPG-coated substrate on cell adhesion, neuronal migration and neurite outgrowth. These results indicate that the C4S-binding peptides neutralize several inhibitory functions of CSPGs, suggesting that they may be beneficial in repairing mammalian nervous system injuries. 3427 KHSHLGSFERHL(3/24) HSPQYWVHHWRG(2/24) SPHSLKHHFHPV(1/24) NNPWHSLHSHTY(1/24) NPGHIGHRHHHT(1/24) WPYTRTHVHHVP(1/24) APHLKHHGLSFR(1/24) AWRHNHFTPVAQ(1/24) VGFHHLTEHPHR(1/24) RLQHQHFHPHVL(1/24) HLKIHHVRVDHM(1/24) TVRHHTHEVSNL(1/24) TYTHHSSSQHYG(1/24) LGSSHGHGASHQ(1/24) FHHH*TQRPAQG(1/24) 15 Phage display (subtractive panning) 5 PMID:30496775 Receptor tyrosine-protein kinase erbB-2 Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) Biopanning was performed as follows: 1. Immobilization of HER2 to Ni-NTA magnetic particles (MPs); 2. Blocking with BSA; 3. Phage binding; 4. Washing and elution of bound phage; 5. Amplification; 6. Negative selection (against BSA-blocked MPs). 3428 GQSEHHMRVASF(60/67)[0.42] GLHTSATNLYLH(3/67)[0.76] STPIFAEATARS(2/67)[1.20] SGVYKVAYDWQH(1/67)[0.33] ALKTHSVSPAPR(1/72)[1.11] 5 Phage display (subtractive panning) 5 PMID:30496775 Receptor tyrosine-protein kinase erbB-2 Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA The absorbance at 450 nm was measured after the reaction with the HRP-anti-M13 antibody conjugate (diluted 1:2000 in the blocking buffer). A450 was reproduced from Fig. 2 and shown. Biopanning was performed as follows: 1. Immobilization of HER2 to Ni-NTA magnetic particles (MPs); 2. Phage binding; 3. Washing and elution of bound phage; 4. Amplification; 5.Two negative selection steps (one against BSA-blocked MPs and another against Ni-NTA MPs). 3429 SGVYKVAYDWQH(37/57)[0.33] HLTTTHPEPPYG(11/57)[1.26] HRGDTTYNHLHP(4/57)[0.88] GQSEHHMRVASF(1/57)[0.42] YSHTLKIPAPDF(1/57)[1.43] 5 Phage display (subtractive panning) 5 PMID:30496775 Receptor tyrosine-protein kinase erbB-2 Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA The absorbance at 450 nm was measured after the reaction with the HRP-anti-M13 antibody conjugate (diluted 1:2000 in the blocking buffer). A450 was reproduced from Fig. 2 and shown. Biopanning was performed as follows: 1. Binding between HER2 and phages; 2. Immobilization of HER2-phage binding mixture to Ni-NTA magnetic particles (MPs); 3. Washing and elution of bound phage; 4. Two negative selection steps (one against BSA-blocked MPs and another against Ni-NTA MPs). 3430 TPGGFAAVG TPGGFGLEL TPGGFLEIV TPGGFWGWM GPVGFSGLE GGSLFRGVV GSALFGIHH CSELFIQHH RVCLFMDMG EMSAFSSHH SYYAFSLVN PGGIFWFSL SVSFFMTHH VRWYFHHHH GTMYFLGGK GTMYFLGGK GTLMFLQRV TVSQFLSHH PGGSFLPLV GTVSFLTGN SVLSFMVGT DIIEFVGVH NEARFADQH GVVYFRGEA VPIDFRVPG GWESFKSLL EVSTFETEE YWALYRHHH VCELTTQEH QLGLYEFVV TPGGYLDYS VTLGYGVEH TPGGYSSMW SVLPYVTHH EDVPYSAHH VLEPYWSAQ GGEAYLSIG GGTVYWDVP RVGVYQEVS LCWQYSPHH YYTFYHHHH ESDFYSTHH SMMRYGEGM GPARYNSVL PGGMYSGTS HKADYSISA GAVVWGEGL SGSVWLDHH GAEVWMDAT GGLVWSYYI GRSVWSFMF HVAVWYAGH GGPLWAEGF GMNLWFAQD GWSLWFAGG GPVAWWYYP DIFAWHHHH PLAGWGHHH LSSFWNGEH RVISWMLPH PGGNWVSYS LVETWLTRP GTIEWAIRV 63 Phage display (common panning) 5 PMID:30458129 Cysteine protease CP16160 Not determined. Not determined. Not determined. X9 T7 phage display library Briefly, an aliquot of amplified phages (e9 pfu) were firstly bound to 125 μl of Ni–nitriloacetic acid (Ni–NTA) beads by their His6-tags for 1 h at 4 °C under gentle agitation. Unbound phages were removed by washing 10 times in 1.5 ml of 1M NaCl, 0.1% Tween-20 in PBS, pH 7.2 and two subsequent washes with 1.5 ml PBS. The Ni-NTA beads were then resuspended in 375 μl PBS. The purified CP (CP16160, ~800 ng) was added to the resuspended Ni-NTA beads to digest the susceptible phage nanopeptides under gentle agitation at 37 °C for 2 h. PBS without added enzyme was used as control. After 2 h incubation, phages with random peptide that was susceptible to protease cleavage were released from Ni-NTA matrix, and the supernatant containing these phages was recovered. To ensure that all of the released phages were recovered, the beads were re-suspended in 100 μl PBS (pH 7.2) and the supernatant, after mixing and centrifugation, was added to the first supernatant. To ensure that the His6-tags had been hydrolyzed on all phages recovered after protease digestion, 15 μl fresh Ni–NTA agarose beads were added to the combined phage supernatant and the mixture agitated for 15 min followed by centrifugation. A control elution of the phages still bound to the beads, using 100 μl 100 mM imidazole showed that at least 1e8 phages were attached to the matrix during each selection. 10 μl of the supernatant containing the released phages were used to determine the amount of phages detached in each round of selection. Dilutions of the supernatant were plated in 2.5 ml of 0.6% top agarose containing 300 μl of Escherichia coli (BLT5615), 100 μl diluted supernatant and 100 μl 100 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). The remaining volume of the supernatant was added to a 10 ml culture of BLT5615 (OD ~0.6). The bacteria had 30 min prior to phage addition been induced to produce the T7 phage capsid protein by the addition of 100 μl 100 mM IPTG to the culture. The bacteria were lysed ~75 min after phage addition. The lysate was centrifuged to remove cell debris and 500 μl of the phage sub-library was added to 100 μl fresh Ni–NTA beads to start the next round of selection. After binding of the sub-library for 1 h at 4 °C under gentle agitation, the Ni–NTA beads were washed 15 times in 1.5 ml 1M NaCl, 0.1% Tween-20 in PBS, pH 7.2, followed by two subsequent washes with 1.5 ml PBS. 3431 ACPMNESKFC(12/22)[29.1] ACPSNPSKFC(6/22)[32.1] ACPKNPNKFC(2/22)[31.4] 3 Phage display (common panning) 3 PMID:30262856 Natalizumab Not determined. Not determined. Not determined. Ph.D.-C7C phage display library (CX7C) Biolayer interferometry (BLI) To determine binding constants for association and dissociation of natalizumab with Ntz-01–07, biolayer interferometry (BLI) was performed by Antibody Solutions (Mountain View CA), using an Octet Red96 (Pall ForteBio LLC, Fremont CA). Briefly, 10 μg/ml of peptide was immobilized onto a streptavidin sensor. Three-fold dilutions of natalizumab were in solution, at concentrations from 1–600 nM (7 total dilutions). The assay steps were: sensor check (30 s), load antibody (600 s), baseline (300 s), antibody association (550 s), antibody dissociation (550 s). TBS was used as the buffer. KD (nM) for synthesized peptides was determined and shown. PKNPSKF is identified as a novel natalizumab-binding motif, and peptides containing this motif demonstrated utility as capture reagents in enzyme-linked immunosorbent assays (ELISAs). A peptide containing the identified motif was shown to be competitive with the natural ligand (α4-integrin) and a neutralizing anti-idiotype antibody for natalizumab binding. 3432 IYAAYPPCPQNLSKFCRHSSSPG(6/20)[NA] AYPHGRSCPQNISKFCFDHEKTN(5/20)[6.03] MPSPPKNCSKFHSALCKGVTWNV(2/20)[NA] SHPQEFWCPQNFSKFCSRSYSNT(1/20)[10.5] QGGEWHRCMSEEGKHCVDIQFIR(1/20)[NA] TSLTVMTCPHNPSKWCSPLPAAV(1/20)[NA] AMASSATCTKPNSYSCLHAKLVP(1/20)[NA] 7 Phage display (common panning) 3 PMID:30262856 Natalizumab Not determined. Not determined. Not determined. X7CX7CX7 M13 phage display library (X7CX7CX7) Biolayer interferometry (BLI) To determine binding constants for association and dissociation of natalizumab with Ntz-01–07, biolayer interferometry (BLI) was performed by Antibody Solutions (Mountain View CA), using an Octet Red96 (Pall ForteBio LLC, Fremont CA). Briefly, 10 μg/ml of peptide was immobilized onto a streptavidin sensor. Three-fold dilutions of natalizumab were in solution, at concentrations from 1–600 nM (7 total dilutions). The assay steps were: sensor check (30 s), load antibody (600 s), baseline (300 s), antibody association (550 s), antibody dissociation (550 s). TBS was used as the buffer. KD (nM) for synthesized peptides was determined and shown. 3433 QTLNHSWLHTFI(1/9) 1 Phage display (common panning) 3 PMID:30262856 Natalizumab Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) 3434 CSDRIMRGC[13.20] 1 Phage display (in vivo) 3 PMID:30082470 Human bladder cancer cell line RT112 Not determined. Not determined. Not determined. Ph.D.-C7C phage display library (CX7C) ELISA For ELISA, RT112 and human normal urothelial cell line SV-HUC-1SV-HUC-1 were plated at 1.0e4 cells/well in a 96-well plate and incubated at 37°C in 5% CO2 for 24 hours. Subsequently, cells were washed and fixed with 4% paraformaldehyde for 15 minutes at room temperature. After incubation with 3% H2O2 (100 mL/well) at 37°C for 30 minutes, the plates were washed 3 times with PBS, and the wells were blocked with 200 mL of 5% BSA for 30 minutes at 37°C. The phages (1.0e10 PFU/well) were added to RT112 or SV-HUC-1 cells and incubated at 37°C for 2 hours. The plates were then washed 3 times with TBST before the addition of 100 mL of HRP-anti-M13 mAb (1:5,000) for 1 hour at 37°C. After washing 3 times with 0.05% TBST, 3,30,5,50-tetramethylbenzidine (TMB) was added at room temperature for 30 minutes. The reaction was terminated by the addition of 50 mL of 2 mol/L H2SO4. Subsequently, absorbance was read using an automated ELISA plate reader at 450 nm. Unrelated phages with equal titers were added to the wells in place of selected phage clones to serve as negative controls. Selectivity was determined using the following formula: Selectivity = [RT112(OD450 - OD450negative control)]/[SV-HUC-1(OD450 - OD450negative control)]. The selectivity value was reproduced from Figure 1A and shown. Subcutaneous tumor-bearing mice received 3e10 plaque-forming units (PFU) of the phage peptide library via tail-vein injection. After 15 minutes, mice were sacrificed and perfused by injection of 50 mL of PBS through the heart to wash unbound phages. Then, a portion of tumors was harvested and preserved in 4% paraformaldehyde and the rest was manually homogenized and washed with 10 × TBS containing 0.1% Tween-20 (0.1% TBST) to remove nonspecifically bound phages. Cell membrane–bound phages were eluted with 1 mL of elution buffer (0.2 mol/L glycine-HCl, pH 2.2, 1 mg/mL BSA) for 10 minutes on ice and neutralized with 150 mL of 1 mol/L Tris–HCl (pH 9.0). After centrifugation, the supernatant was collected, and the cells in the precipitate were washed once with PBS-BSA and lysed with 0.1% Triton X-100 for 2 hours at room temperature. Thus, the internalized phages in the cell lysate were recovered. A total of 10 mL of eluted phage was titrated on agar plates in the presence of IPTG/X-gel (1 mg/L) and tetracycline (40 mg/mL). The remaining phages were amplified by ER2738 bacteria at 37°C for 4.5 hours and injected into tumor-bearing mice. The biopanning process was repeated for three rounds. A bladder cancer–specific peptide with the sequence of CSDRIMRGC (PLSWT7) was selected by in vivo phage-display technology and labeled with IRDye800CW to synthesize a bladder cancer–specific dual-modality imaging (DMI) probe (PLSWT7-DMI). The feasibility of PLSWT7-DMI–based dual-modality photoacoustic imaging near-infrared (PAI-NIR) imaging was assessed in vitro, in mouse models, and ex vivo human bladders. An air-pouch bladder cancer (APBC) model suitable for probe instillation was established to evaluate the probe-based bladder cancer PAI diagnosis and NIR-imaging–guided resection. Human bladders were used to assess whether the PLSWT7-DMI–based DMI strategy is a translatable approach for bladder cancer detection and resection. The probe exhibited excellent selectivity and specificity both in vitro and in vivo. Postinstillation of the probe, tumors <3mm were detectable by PAI, and NIR-imaging–guided tumor resection decreased the bladder cancer recurrence rate by 90% and increased the survival in the mouse model. Additionally, ex vivo NIR imaging of human bladders indicated that PLSWT7-DMI–based imaging would potentially allow precise resection of bladder cancer in clinical settings. This PLSWT7-DMI–based DMI strategy was a translatable approach for bladder cancer diagnosis and resection and could potentially lower the bladder cancer recurrence rate. 3435 CNWMINKEC(3/20)[0.72 ± 0.09] CVPSKPGLC(3/20)[0.21 ± 0.02] CPKGDENTC(2/20)[0.12 ± 0.02] CPTSQRDNC(2/20)[0.16 ± 0.01] CVEKSAMSC(1/20)[NA] CLSTTEGYC(1/20)[NA] CIHSPTALC(1/20)[NA] CSLASTSHC(1/20)[NA] CLKPHSADC(1/20)[NA] CLHKSVSGC(1/20)[NA] CSSKHEATC(1/20)[NA] CSKMKIDHC(1/20)[NA] CSENSPLLC(1/20)[NA] CNQTEPFSC(1/20)[NA] 14 Phage display (subtractive panning) 3/4/5 PMID:30282451 Hepatitis A virus cellular receptor 1, HAVcr-1 Not determined. Not determined. Not determined. Ph.D.-C7C phage display library (CX7C) ELISA Individual phage clones were incubated with KIM-1‒coated plates and the bound phage clones were determined by ELISA. Plates were read at 450 nm using a microplate reader (Tecan, Zurich, Switzerland). The absorbance at 450 nm represent as the mean±SD of three separate experiments performed in triplicate, which was reproduced from Figrue 2A and shown. The absorbance of the wild type M13 phage was 0.10 ± 0.01. Each biopanning round consisted of subtraction of phages nonspecifically bound to BSA and subsequent selection of phages bound to KIM-1, followed by amplification of the eluted phages. A phage clone displaying the CNWMINKEC peptide showed higher binding affinity to KIM-1 and KIM-1-overexpressing 769-P renal tumor cells compared to other phage clones selected after biopanning. The CNWMINKEC peptide and a NeutrAvidin/biotin-CNWMINKEC multimer selectively bound to KIM-1 over albumin and to KIM-1-overexpressing 769-P cells and A549 lung tumor cells compared to KIM-1-low expressing HEK293 normal cells. Co-localization and competition assays using an anti-KIM-1 antibody demonstrated that the binding of the CNWMINKEC peptide to 769-P cells was specifically mediated by KIM-1. The CNWMINKEC peptide was not cytotoxic to cells and was stable for up to 24 hours in the presence of serum. Whole-body fluorescence imaging demonstrated selective homing of the CNWMINKEC peptide to KIM-1‒overexpressing A498 renal tumor compared to KIM-1‒low expressing HepG2 liver tumor in mice. 3436 YHMEFWDELWGI(4)[0.55 ± 0.11] WHNPLWWLSAYE(3)[0.61 ± 0.09] WHEYPLVWLSGY(3)[0.47 ± 0.14] 3 Phage display (common panning) 4 PMID:30269789 Glycoprotein 4, Protein GP4 Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA The binding activities between the selected phages and GP4 recombinant protein were determined by phage ELISA. OD405 was detected with an ELISA plate reader. The original mixed phage library was used as the negative control. The OD405 value was reproduced from Figure 2A and shown. Phage display biopanning on GP4 protein showed that the specific phages obtained could distinguish porcine reproductive and respiratory syndrome virus (PRRSV) from the other viruses. The exogenous peptide WHEYPLVWLSGY displayed on one of the candidate phages showed high affinity for GP4 protein and exerted a significant inhibitory effect on PRRSV penetration in vitro. Moreover, the N-terminus of GP4 was predicted as the critical receptor binding site and the beginning of the fifth scavenger receptor cysteine-rich domain of CD163 as the critical ligand recognition site based on sequence alignment and model prediction analyses. 3437 GLHTSATNLYLH(13)[12.6] SGVYKVAYDWQH(6)[5.7] FIPFDPMSMRWE(3)[7.2] ALAVAPSRWWNE(3)[76.3] KASGSPSGFWPS(3)[NA] NASSFPTNSRWA(2)[20.6] QFDYMRPANDTH(2)[3.3] GQSEHHMRVASF(2)[NA] GTGLVTLPRLTV(2)[NA] DLILLNGGSQQA(1)[NA] LPVGNQSDVHFD(1)[NA] SPLCCNSGTDQT(1)[NA] DPIKHLEDQAST(1)[NA] AAIEPSAGLAYF(1)[NA] SPYTWGSGDWRS(1)[NA] DQFVHDVKGTKH(1)[NA] AHWGMSARTSAW(1)[NA] TNRTHNINALHF(1)[NA] GNHNNSVTTWHL(1)[NA] MHPNAGHGSLMR(1)[NA] TPGGLLRNDRPL(1)[NA] NIYTTPWGSNWS(1)[NA] HLARTTDMLHPY(1)[NA] QMSPGSTARSAL(1)[NA] TVQSLPPHSPYD(1)[NA] MDNKTTRWRDPL(1)[NA] HSIALTTTLRTP(1)[NA] MSKSTMSLSSNA(1)[NA] TSIQISNAHPKS(1)[NA] HTSYGTTWSESR(1)[NA] NHLSTPVWSITG(1)[NA] WSMNHTNLSYTS(1)[NA] QPDFTPRPAYSR(1)[NA] ITGCDPTKQTQG(1)[NA] AYPQKFNNNFMS(1)[NA] YDIHWSPDSRLK(1)[NA] ASATVSKPWPTG(1)[NA] KVMGLSVTSHAE(1)[NA] SNSSLELPNLNS(1)[NA] YIWQGPGSAGTP(1)[NA] HGNPRSPNQFST(1)[NA] QCFNSVCLHTNP(1)[NA] QQHPLITRLISH(1)[NA] ALEVNGMRPAWA(1)[NA] HRGDTTYNHLHP(1)[NA] SQGLSLSTMVTR(1)[NA] ADPLGISYLRSS(1)[NA] KATDFPDGRLNY(1)[NA] SAELPHKTGSVL(1)[NA] GSAPLLTVDTSK(1)[NA] STPIFAEATARS(1)[NA] LPHLPSQNVRAP(1)[NA] SCDMQERCLAGL(1)[NA] VLYEQVLAAPME(1)[NA] TAGAIADFQSLT(1)[NA] LTHYPVTGSTGS(1)[NA] SEFQSTHWLDSL(1)[NA] WIRPPSGPMYSF(1)[NA] DPTRQEPHLFLL(1)[NA] QAMMWLPVALAA(1)[NA] SGKLLMLPVVSR(1)[NA] YTTIRWSEFSAW(1)[NA] DRWVARDPASIF(1)[NA] ISSTDRLLAAGL(1)[NA] SGTSLLHYHSYH(1)[NA] DSQFNKYSIATV(1)[NA] SNLSARNTISHG(1)[NA] MDGSSREVNDAV(1)[NA] 68 Phage display (common panning) 4 PMID:29956504 Human breast cancer cell line MCF7 Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) Flow Cytometry The fluorescence intensity of each FITC-labeled peptide at 1.0e–6 m (also defined as the fluorescence per unit, FPU) was measured by a spectrofluorometer (VARIAN, Cary Eclipse, USA, Ex = 488 nm, Em = 535 nm). The FPU was used for calibration of the relative affinity. The mean fluorescence intensity (MFI) of 10000 cells was measured by a FACSCalibur flow cytometer (Becton Dickinson, USA). MCF7 cells incubated with blank RPMI-1640 medium were taken as the background group and MCF7 cells incubated with FITC-AGP were used as the control group. The relative affinity of a peptide to MCF7 cells was calculated using the following formula: Relative affinity = ((MFIpeptide -MFIbackground)/FPUpeptide)/((MFIcontrol - MFIbackground)/FPUcontrol), which means the affinity ratio of the given peptide to the control peptide. The mean relative affinity of ligand peptides to MCF7 cells was shown in Figure 2A and shown. MCF7 cells were seeded in a 25 cm2 flask and cultured until confluence (about 3 .0e6 cells per flask). Then the cells were rinsed twice with PBS and blocked with 0.5% bovine serum albumin in RPMI-1640 medium (BSA-1640) at 4 °C for 30 min. After blocking, the cells were coincubated with phages (1.0e11 pfu, Ph.D.-12 peptide library kit, the input phage library, displaying a diversity of 1.0e9 dodecapeptides) in BSA-1640 at 4 °C for 30 min to allow for binding or internalization between phages and cells. The medium containing unbound phages was discarded and the cells were washed ten times with BSA-1640 containing 0.1% Tween-20 to remove weakly bound phages. Afterward, the phages that bound strongly to MCF7 cells were eluted down with Glycine-HCl (2 m, pH 2.2) solution and the acidic eluate was recovered as one output phage sublibrary, which had higher cell-binding affinity to MCF7 cells than the input phage library. Finally, after washing thrice, the cells were lysed by Tris-EDTA solution (50e−3 m Tris, 1.0e−3 m EDTA, 150e−3 m NaCl, 2% deoxycholate, pH 10.0) and the cell lysate containing intracellular phages was recovered as another output phage sublibrary, which had higher cell-internalizing efficacy to MCF7 cells than the input phage library. The phage titer was assayed by counting plague forming units. The recovery rate of phage in each round was calculated as the ratio of output to input. The two output phage sublibraries (eluate and lysate) were amplified and used as the input phage libraries of the next panning round, respectively. A total of four rounds were performed to achieve phage sublibraries with sufficient affinity to MCF7 cells. Four of these ligand peptides [FIPFDPMSMRWE (FIP), NASSFPTNSRWA (NAS), GLHTSATNLYLH (GLH), and ALAVAPSRWWNE (ALA), respectively] exhibit high affinity to MCF7 human breast cancer cells. Among them, NAS and ALA are reported for the first time, whose affinities are 20.6 and 76.3 times that of the random peptide control, respectively. Both NAS and ALA modifications to doxorubicin-loaded lipid nanosytems [LP(DOX)] show stronger tumor inhibition, longer animal survival time, and less body weight loss, compared to unmodified or control peptide modified nanosystems, on an MCF7 tumor-bearing mouse model. 3438 CYLLCISPC CGSRCYPRC 2 Phage display (subtractive panning) 1 PMID:30198459 IgG from tegumentary leishmaniasis (TL) patients Not determined. Not determined. Not determined. Ph.D.-C7C phage display library (CX7C) Briefly, viral particles of the bacteriophage library (Ph.D.®-C7C library, New England BioLabs, Ipswich, MA, USA) were diluted in 250 μL of 50mM Tris-HCl pH 7.5, 150 mM NaCl and 0.1% Tween 20 buffer (TBS-T). The mixture was incubated for 30 min, at room temperature, with microspheres coupled to the IgG antibodies purified from healthy subjects for subtraction, which were magnetically captured. The remaining phages in the supernatant were recovered and transferred to a new tube, which was subjected to positive selection using IgG from TL patients. The supernatant was removed and the bound phages were washed five times in 1 mL TBS-T buffer, and were eluted in 500 μL of 0.2 M glycine buffer, pH 2.0. Next, 75 μL of 1 M Tris-base pH 9.0 were added to neutralize the acidic pH of the solution. Two clones, namely A4 (CYLLCISPC) and A8 (CGSRCYPRC), were identified and used in immunization protocols from BALB/c mice to protect against Leishmania amazonensis infection. Results showed a polarized Th1 response generated after vaccination, being based on significantly higher levels of interferon-γ (IFN-γ), interleukin-2 (IL-2), IL-12, tumour necrosis factor-α (TNF-α) and granulocyte-macrophage colony-stimulating factor (GM-CSF); which were associated with lower production of specific IL-4, IL-10 and immunoglobulin G1 (IgG1) antibodies. Vaccinated mice presented significant reductions in the parasite load in the infected tissue and distinct organs, when compared with controls. 3439 GWIVSFAVQTEGHGVAD(70) GGTQCLQFKVIPAGHFC(40) GIARSNQDIFVYDAHKC(9) GYYPSVGHQTCQDIFVA(8) GYIVSQTEGRHPQHQEA(4) GAGFCYQQYLEQDIFVY(2) GWKNCAVVQTEGCNCYF(2) GSIWSQVQTEGYRHNHA(2) GEELCQTEGRGPEVQPY(2) GNLACWYNEQDIFVAHD(1) GVFGSITVVQTEGVYSC(1) GTWSCRYYQQVQTEGQD(1) GVNVSVQTEGAWHAQAY(1) GVHMCKLHLLVQTEGQC(1) GQPTCYNHNKQQTEGRC(1) 15 Phage display (competitive panning) 2 PMID:30267550 Serum 13003 from patients sensitized to soy bean Not determined. Not determined. Not determined. ENTE-1 phage display library (GX3[C/S]X12) A trinucleotide‐based, 16mer peptide phagemid library covering >e9 clones (“ENTE‐1”) was used for two rounds of panning against individual serum from patients sensitized to soy bean. We coated 24 μg total serum protein in 2.5 mL PBS to Immuno Tubes (MaxiSorp™; Thermo Fisher Scientific, Waltham, MA, USA) for 1 hour at 4°C under constant agitation. Under these conditions, all Ig molecules should be bound on the tube surface. A negative control was incubated with PBS only. The coated tubes were washed with 2.5 mL blocking buffer once (5% [w/v] non‐fat dry milk powder in PBS) and incubated with 4 mL blocking buffer at 4°C for at least 15 minutes under constant agitation. Before adding the ENTE‐1 peptide phage display library, the tubes were washed three times with 4 mL PBS. To preferably select patient-specific antibodies, and in particular to prevent selection of peptides binding serum proteins, a competition with a mixture of ten sera from healthy donors was conducted. The ENTE‐1 library was added to each tube in 1 mL blocking buffer containing 30 μg of non‐allergic serum protein. In the first selection round, an amount of phage corresponding to 4e11 colony forming units (cfu) of ENTE‐1 library and in the second selection only e9‐e10 cfu of recovered phage were used. Tubes were incubated for 2 hour at 4°C with constant agitation, washed five times with 1 mL 0.1% (v/ v) Tween 20 in PBS in the first selection round, and with 0.5% (v/v) Tween 20 in PBS in the second selection round. 3440 GYQWSTEHIFVIPCQRA(4) GEPQSEQDIFQKCEKQF(1) GTFHSIQQQRDUFVIVI(1) 3 Phage display (competitive panning) 2 PMID:30267550 Serum 14008 from patients sensitized to soy bean Not determined. Not determined. Not determined. ENTE-1 phage display library (GX3[C/S]X12) A trinucleotide‐based, 16mer peptide phagemid library covering >e9 clones (“ENTE‐1”) was used for two rounds of panning against individual serum from patients sensitized to soy bean. We coated 24 μg total serum protein in 2.5 mL PBS to Immuno Tubes (MaxiSorp™; Thermo Fisher Scientific, Waltham, MA, USA) for 1 hour at 4°C under constant agitation. Under these conditions, all Ig molecules should be bound on the tube surface. A negative control was incubated with PBS only. The coated tubes were washed with 2.5 mL blocking buffer once (5% [w/v] non‐fat dry milk powder in PBS) and incubated with 4 mL blocking buffer at 4°C for at least 15 minutes under constant agitation. Before adding the ENTE‐1 peptide phage display library, the tubes were washed three times with 4 mL PBS. To preferably select patient-specific antibodies, and in particular to prevent selection of peptides binding serum proteins, a competition with a mixture of ten sera from healthy donors was conducted. The ENTE‐1 library was added to each tube in 1 mL blocking buffer containing 30 μg of non‐allergic serum protein. In the first selection round, an amount of phage corresponding to 4e11 colony forming units (cfu) of ENTE‐1 library and in the second selection only e9‐e10 cfu of recovered phage were used. Tubes were incubated for 2 hour at 4°C with constant agitation, washed five times with 1 mL 0.1% (v/ v) Tween 20 in PBS in the first selection round, and with 0.5% (v/v) Tween 20 in PBS in the second selection round. 3441 GSENCDIFVIQEIQSCF(3) GLNRCDIFVIPECELHN(2) 2 Phage display (competitive panning) 2 PMID:30267550 Serum 13009 from patients sensitized to soy bean Not determined. Not determined. Not determined. ENTE-1 phage display library (GX3[C/S]X12) A trinucleotide‐based, 16mer peptide phagemid library covering >e9 clones (“ENTE‐1”) was used for two rounds of panning against individual serum from patients sensitized to soy bean. We coated 24 μg total serum protein in 2.5 mL PBS to Immuno Tubes (MaxiSorp™; Thermo Fisher Scientific, Waltham, MA, USA) for 1 hour at 4°C under constant agitation. Under these conditions, all Ig molecules should be bound on the tube surface. A negative control was incubated with PBS only. The coated tubes were washed with 2.5 mL blocking buffer once (5% [w/v] non‐fat dry milk powder in PBS) and incubated with 4 mL blocking buffer at 4°C for at least 15 minutes under constant agitation. Before adding the ENTE‐1 peptide phage display library, the tubes were washed three times with 4 mL PBS. To preferably select patient-specific antibodies, and in particular to prevent selection of peptides binding serum proteins, a competition with a mixture of ten sera from healthy donors was conducted. The ENTE‐1 library was added to each tube in 1 mL blocking buffer containing 30 μg of non‐allergic serum protein. In the first selection round, an amount of phage corresponding to 4e11 colony forming units (cfu) of ENTE‐1 library and in the second selection only e9‐e10 cfu of recovered phage were used. Tubes were incubated for 2 hour at 4°C with constant agitation, washed five times with 1 mL 0.1% (v/ v) Tween 20 in PBS in the first selection round, and with 0.5% (v/v) Tween 20 in PBS in the second selection round. 3442 GSWPSLEIFVIPIFVQI(112) 1 Phage display (competitive panning) 2 PMID:30267550 Serum 13007 from patients sensitized to soy bean Not determined. Not determined. Not determined. ENTE-1 phage display library (GX3[C/S]X12) A trinucleotide‐based, 16mer peptide phagemid library covering >e9 clones (“ENTE‐1”) was used for two rounds of panning against individual serum from patients sensitized to soy bean. We coated 24 μg total serum protein in 2.5 mL PBS to Immuno Tubes (MaxiSorp™; Thermo Fisher Scientific, Waltham, MA, USA) for 1 hour at 4°C under constant agitation. Under these conditions, all Ig molecules should be bound on the tube surface. A negative control was incubated with PBS only. The coated tubes were washed with 2.5 mL blocking buffer once (5% [w/v] non‐fat dry milk powder in PBS) and incubated with 4 mL blocking buffer at 4°C for at least 15 minutes under constant agitation. Before adding the ENTE‐1 peptide phage display library, the tubes were washed three times with 4 mL PBS. To preferably select patient-specific antibodies, and in particular to prevent selection of peptides binding serum proteins, a competition with a mixture of ten sera from healthy donors was conducted. The ENTE‐1 library was added to each tube in 1 mL blocking buffer containing 30 μg of non‐allergic serum protein. In the first selection round, an amount of phage corresponding to 4e11 colony forming units (cfu) of ENTE‐1 library and in the second selection only e9‐e10 cfu of recovered phage were used. Tubes were incubated for 2 hour at 4°C with constant agitation, washed five times with 1 mL 0.1% (v/ v) Tween 20 in PBS in the first selection round, and with 0.5% (v/v) Tween 20 in PBS in the second selection round. 3443 GIRACWQAPFVIPAGIC(1) 1 Phage display (competitive panning) 2 PMID:30267550 Serum 13016 from patients sensitized to soy bean Not determined. Not determined. Not determined. ENTE-1 phage display library (GX3[C/S]X12) A trinucleotide‐based, 16mer peptide phagemid library covering >e9 clones (“ENTE‐1”) was used for two rounds of panning against individual serum from patients sensitized to soy bean. We coated 24 μg total serum protein in 2.5 mL PBS to Immuno Tubes (MaxiSorp™; Thermo Fisher Scientific, Waltham, MA, USA) for 1 hour at 4°C under constant agitation. Under these conditions, all Ig molecules should be bound on the tube surface. A negative control was incubated with PBS only. The coated tubes were washed with 2.5 mL blocking buffer once (5% [w/v] non‐fat dry milk powder in PBS) and incubated with 4 mL blocking buffer at 4°C for at least 15 minutes under constant agitation. Before adding the ENTE‐1 peptide phage display library, the tubes were washed three times with 4 mL PBS. To preferably select patient-specific antibodies, and in particular to prevent selection of peptides binding serum proteins, a competition with a mixture of ten sera from healthy donors was conducted. The ENTE‐1 library was added to each tube in 1 mL blocking buffer containing 30 μg of non‐allergic serum protein. In the first selection round, an amount of phage corresponding to 4e11 colony forming units (cfu) of ENTE‐1 library and in the second selection only e9‐e10 cfu of recovered phage were used. Tubes were incubated for 2 hour at 4°C with constant agitation, washed five times with 1 mL 0.1% (v/ v) Tween 20 in PBS in the first selection round, and with 0.5% (v/v) Tween 20 in PBS in the second selection round. 3444 GSAVCSYGFVIPAGCII(5) GIWGSPAGYPEECVVRD(3) 2 Phage display (competitive panning) 2 PMID:30267550 Serum 14012 from patients sensitized to soy bean Not determined. Not determined. Not determined. ENTE-1 phage display library (GX3[C/S]X12) A trinucleotide‐based, 16mer peptide phagemid library covering >e9 clones (“ENTE‐1”) was used for two rounds of panning against individual serum from patients sensitized to soy bean. We coated 24 μg total serum protein in 2.5 mL PBS to Immuno Tubes (MaxiSorp™; Thermo Fisher Scientific, Waltham, MA, USA) for 1 hour at 4°C under constant agitation. Under these conditions, all Ig molecules should be bound on the tube surface. A negative control was incubated with PBS only. The coated tubes were washed with 2.5 mL blocking buffer once (5% [w/v] non‐fat dry milk powder in PBS) and incubated with 4 mL blocking buffer at 4°C for at least 15 minutes under constant agitation. Before adding the ENTE‐1 peptide phage display library, the tubes were washed three times with 4 mL PBS. To preferably select patient-specific antibodies, and in particular to prevent selection of peptides binding serum proteins, a competition with a mixture of ten sera from healthy donors was conducted. The ENTE‐1 library was added to each tube in 1 mL blocking buffer containing 30 μg of non‐allergic serum protein. In the first selection round, an amount of phage corresponding to 4e11 colony forming units (cfu) of ENTE‐1 library and in the second selection only e9‐e10 cfu of recovered phage were used. Tubes were incubated for 2 hour at 4°C with constant agitation, washed five times with 1 mL 0.1% (v/ v) Tween 20 in PBS in the first selection round, and with 0.5% (v/v) Tween 20 in PBS in the second selection round. 3445 GLVSSQLTQFVIPAHCD(2) 1 Phage display (competitive panning) 2 PMID:30267550 Serum 13002 from patients sensitized to soy bean Not determined. Not determined. Not determined. ENTE-1 phage display library (GX3[C/S]X12) A trinucleotide‐based, 16mer peptide phagemid library covering >e9 clones (“ENTE‐1”) was used for two rounds of panning against individual serum from patients sensitized to soy bean. We coated 24 μg total serum protein in 2.5 mL PBS to Immuno Tubes (MaxiSorp™; Thermo Fisher Scientific, Waltham, MA, USA) for 1 hour at 4°C under constant agitation. Under these conditions, all Ig molecules should be bound on the tube surface. A negative control was incubated with PBS only. The coated tubes were washed with 2.5 mL blocking buffer once (5% [w/v] non‐fat dry milk powder in PBS) and incubated with 4 mL blocking buffer at 4°C for at least 15 minutes under constant agitation. Before adding the ENTE‐1 peptide phage display library, the tubes were washed three times with 4 mL PBS. To preferably select patient-specific antibodies, and in particular to prevent selection of peptides binding serum proteins, a competition with a mixture of ten sera from healthy donors was conducted. The ENTE‐1 library was added to each tube in 1 mL blocking buffer containing 30 μg of non‐allergic serum protein. In the first selection round, an amount of phage corresponding to 4e11 colony forming units (cfu) of ENTE‐1 library and in the second selection only e9‐e10 cfu of recovered phage were used. Tubes were incubated for 2 hour at 4°C with constant agitation, washed five times with 1 mL 0.1% (v/ v) Tween 20 in PBS in the first selection round, and with 0.5% (v/v) Tween 20 in PBS in the second selection round. 3446 GTSQSEAVIPAGHHEHY(15) GEKHSEDPFLVIPAGGD(8) 2 Phage display (competitive panning) 2 PMID:30267550 Serum 13021 from patients sensitized to soy bean Not determined. Not determined. Not determined. ENTE-1 phage display library (GX3[C/S]X12) A trinucleotide‐based, 16mer peptide phagemid library covering >e9 clones (“ENTE‐1”) was used for two rounds of panning against individual serum from patients sensitized to soy bean. We coated 24 μg total serum protein in 2.5 mL PBS to Immuno Tubes (MaxiSorp™; Thermo Fisher Scientific, Waltham, MA, USA) for 1 hour at 4°C under constant agitation. Under these conditions, all Ig molecules should be bound on the tube surface. A negative control was incubated with PBS only. The coated tubes were washed with 2.5 mL blocking buffer once (5% [w/v] non‐fat dry milk powder in PBS) and incubated with 4 mL blocking buffer at 4°C for at least 15 minutes under constant agitation. Before adding the ENTE‐1 peptide phage display library, the tubes were washed three times with 4 mL PBS. To preferably select patient-specific antibodies, and in particular to prevent selection of peptides binding serum proteins, a competition with a mixture of ten sera from healthy donors was conducted. The ENTE‐1 library was added to each tube in 1 mL blocking buffer containing 30 μg of non‐allergic serum protein. In the first selection round, an amount of phage corresponding to 4e11 colony forming units (cfu) of ENTE‐1 library and in the second selection only e9‐e10 cfu of recovered phage were used. Tubes were incubated for 2 hour at 4°C with constant agitation, washed five times with 1 mL 0.1% (v/ v) Tween 20 in PBS in the first selection round, and with 0.5% (v/v) Tween 20 in PBS in the second selection round. 3447 GIQRSAGYPVFKDVDID(4) 1 Phage display (competitive panning) 2 PMID:30267550 Serum 13001 from patients sensitized to soy bean Not determined. Not determined. Not determined. ENTE-1 phage display library (GX3[C/S]X12) A trinucleotide‐based, 16mer peptide phagemid library covering >e9 clones (“ENTE‐1”) was used for two rounds of panning against individual serum from patients sensitized to soy bean. We coated 24 μg total serum protein in 2.5 mL PBS to Immuno Tubes (MaxiSorp™; Thermo Fisher Scientific, Waltham, MA, USA) for 1 hour at 4°C under constant agitation. Under these conditions, all Ig molecules should be bound on the tube surface. A negative control was incubated with PBS only. The coated tubes were washed with 2.5 mL blocking buffer once (5% [w/v] non‐fat dry milk powder in PBS) and incubated with 4 mL blocking buffer at 4°C for at least 15 minutes under constant agitation. Before adding the ENTE‐1 peptide phage display library, the tubes were washed three times with 4 mL PBS. To preferably select patient-specific antibodies, and in particular to prevent selection of peptides binding serum proteins, a competition with a mixture of ten sera from healthy donors was conducted. The ENTE‐1 library was added to each tube in 1 mL blocking buffer containing 30 μg of non‐allergic serum protein. In the first selection round, an amount of phage corresponding to 4e11 colony forming units (cfu) of ENTE‐1 library and in the second selection only e9‐e10 cfu of recovered phage were used. Tubes were incubated for 2 hour at 4°C with constant agitation, washed five times with 1 mL 0.1% (v/ v) Tween 20 in PBS in the first selection round, and with 0.5% (v/v) Tween 20 in PBS in the second selection round. 3448 GTMPDKDTGMRQ ERAGTMPDRPRH GVMLSDRLERAL FQARWEPPRLLQ HSNSYQASVLCQ TLMQPSHAGLTL GGHQVNQLPSQM YSPRLSTLQSTP SSSGNPLYSAKI TLSLPGFTFVPT HTVEPRWVPRSW FLVFLVCTVHPP AVCTFCASDWWP NIWTPSLSLTYD FGDPFVSALFPQ SAPVPGSSHSRQ KPSVPGCTLVPS NCSNSIPCAVKA QAILYSPDRNPL TITNAPIKDLTP QVNGLGERSQQM TLQPQSRDLSTL YLTPELFLRTQQ MLQDNLQNLDFR LNPNWTYRSTNE HEASRLLGARLT VSKAPSKLETY 27 Phage display (common panning) 3 PMID:30187588 IgE from patients with allergy to Scylla paramamosain AK Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) Overnight, coating of the wells of microtiter plate (Nunc Maxisorp, Copenhagen, Denmark) at 4°C with goat anti-human IgE (ε‐chain specific) (Sigma‐Aldrich, St. Louis, MO, USA) was used to capture IgE from patients’ sera. The phage display peptide library was added after the blocking incubation with captured IgE and eluted after the washing steps. 3449 VGSYSATVHRIQ VLAHKYTTGSLP ACLYCYDLVDSV SLPLTVVPTANR TLGLRPVPVATT TNSFHGIAGYQS SSLLGYNLNANH ASHTGTAQISRQ ETLKPYPLPNNS DGMLSLSLPASF VRATQYPMLLDF NGPSDLKYLHPT HPHDRQLISTYP NDVGVLSPETRQ IEQRHQTHSLHP HMSYGNGTRDTP DNHAKLPLRGSS VVFDGTLYNYHW DIARTAKPSHWP AYTVDALHELRH SYEPNSTKQFAP VDDLVRVSVNFR MYNPDPNRLARS WHYNWQDVSDRQ NMGGLDYLNKNS EALTVNIKREME HLTATELANSYH YTLSDRSHNPFN SWIPWAAFEHKQ WTTAAPAKSEYH TPVQQGQTKNAF FMIPGSMPYIGC CLYCWQGTSQQR WDWPLFPLDTLR AYEPSSHKSEVP VPIHAAHGFWHY WPAPFHNLVNPR 37 Phage display (common panning) 3 PMID:30187588 IgE from patients with allergy to Scylla paramamosain SCP Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) Overnight, coating of the wells of microtiter plate (Nunc Maxisorp, Copenhagen, Denmark) at 4°C with goat anti-human IgE (ε‐chain specific) (Sigma‐Aldrich, St. Louis, MO, USA) was used to capture IgE from patients’ sera. The phage display peptide library was added after the blocking incubation with captured IgE and eluted after the washing steps. 3450 SVDGWLEPPTST DIARTAKPSHWP DINIRVYPSQFP LVPSPQSAMRFD AHDSADYLNAPR SHNAPWSPTLAL YPGISHVGSKVS TGSAKFLQRDTH TLMQPSHAGLTL DQYLMTTTRGAP YPGSQSWMPSDF GCNHDSCSALTK DNHAKLPLRGSS TIPTRDPAMLHS 7 Phage display (common panning) 3 PMID:30187588 IgE from patients with allergy to Scylla paramamosain TIM Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) Overnight, coating of the wells of microtiter plate (Nunc Maxisorp, Copenhagen, Denmark) at 4°C with goat anti-human IgE (ε‐chain specific) (Sigma‐Aldrich, St. Louis, MO, USA) was used to capture IgE from patients’ sera. The phage display peptide library was added after the blocking incubation with captured IgE and eluted after the washing steps.