3576 WDMWPSMDWKAE(4/27)[1.0403±0.0736] FGLEPRANLHFT(2/27)[0.6586±0.0164] VQVRDNLPTTTG(2/27)[0.2498±0.0172] THDMGKMDRTTT(2/27)[0.307±0.0265] WGNSQWTGQHTT(1/27)[0.7087±0.0236] 5 Phage display (subtractive panning) 2-3 PMID:35168735 Interleukin-33 (IL-33) Interleukin-1 receptor-like 1 Not determined. Not determined. Ph.D.-7 phage display library (X7) ELISA ELISA was performed to evaluate the binding affinity of the phage particles to IL-33. First, streptavidin-coated wells were incubated with 100 μL of biotinylated IL-33 (1 μM) at room temperature for 1 h. The supernatant with residual proteins or unbound phages was subsequently discarded, and then, 200 μL of blocking buffer comprising 0.1 M NaHCO3 (pH 8.6) with 5 mg/mL of BSA and 0.02% NaN3, was added to the wells and incubated for 1 h at 4 °C. The wells were filled with blocking buffer. After incubation, the plate wells were washed 6 times with 0.5% PBST (0.1 M PBS (pH 7.4) with 0.5% Tween 20). The screened peptide-displaying phages (1 × 1011 or 1 × 1012 pfu/mL) were added to the pre-functionalized wells and incubated for 1 h at room temperature with mild shaking. The phage solutions were removed, and then the wells were washed six times with 0.5% PBST to remove unbound phages. The wells were then filled with HRP-conjugated anti-M13 monoclonal antibodies (diluted 1:5000 in blocking buffer) and incubated for 1 h at room temperature with mild shaking. Finally, the wells were washed with 0.5% PBST and filled with 200 μL ABTS (with H2O2), which is an HRP substrate. The change of absorbance was measured at 405 nm using a microplate spectrophotometer (Multiskan FC, Thermo Scientific, Waltham, MA, USA). The change of absorbance at 405 nm was reproduced from Figure 1A. We performed three rounds of biopanning against IL-33 protein. A negative selection beginning with the 2nd round, in which the amplified phage was preincubated with the streptavidin-coated plate in the absence of IL-33, was performed. The selected IL-33 specific peptide was identified as FGLEPRANLHFT. To investigate the molecular interactions between IL-33 and the affinity peptide, the peptide was separated from the phage particles, chemically synthesized and characterized by square wave voltammetry (SWV), isothermal titration calorimetry (ITC), and microscale thermophoresis (MST). The binding constant (Kd) value with SWV, MST, and ITC was found to be 1.68 ± 0.37 μM, 5.98 ± 1.30 μM, and 2.68 ± 1.37 μM, respectively. Two-dimensional (2D) NMR spectral analysis was performed to elucidate the primary peptide binding site of IL-33, which was near the ST2-D3 and IL1RAcP-D3 binding interfaces. 3577 CPMKSHTNC(4) CQGTSLSHC(1) CDQRLPNYC(1) CITNHSPTC(1) CPTGPSATC(1) CTSSEPNLC(1) CNSSRSELC(1) 7 Phage display (in vivo) 3 PMID:34185171 Cerebrospinal fluid Not determined. Not determined. Not determined. Ph.D.-C7C phage display library (CX7C) To obtain the targeting peptides, the appropriate time to recover phages from the CSF was determined using a recovery time curve. The titer (in transducing units/ml CSF) of the phages recovered was plotted against time to construct the recovery time curve (Li et al. 2015). Adult male Sprague–Dawley rats were used for three rounds of screening. In the first round, Sprague–Dawley rats (n = 3) were injected intravenously (i.v.) with 1012 pfu of Ph.D.-C7C™ phage display library suspended in 100 μl of tris-buffered saline (TBS) (50 mM Tris–HCl, 150 mM NaCl, pH 7.5). The phages were allowed to circulate in the rats for 1 h before CSF was drawn. Sprague–Dawley rats were anesthetized using 5% chloral hydrate (0.4 g/kg). Phages recovered from the CSF were then incubated with E. coli (ER2738 host strain) for amplification. The other two rounds of biopanning were proceeded by injecting rats (i.v.) with newly amplified phages (1 × 1012 pfu in 100 μl TBS) and repeating the subsequent procedures of the first round as described above. A peptide sequence denoted as PMK (CPMKSHTNC), which was demonstrated to be able to cross the blood-cerebrospinal fluid barrier (BCSFB) via in vivo optical imaging analysis, could be used in the future for the construction of targeted drug delivery systems. 3578 DYHDPSLPTLRK(30.2%)[0.0321±0.0062] QVNGLGERSQQM(15.7%)[NT] RDYHPRDHTATW(7.9%)[NT] GNNPLHVHHDKR(4.0%)[NT] TAKYLPMRPGPL(3.3%)[NT] SPLRAVAFSGAQ(2.4%)[NT] INIVPGPEKPVG(2.2%)[NT] QGYKQEYTRWGE(<0.1%)[NT] 8 Phage display (common panning) 1 PMID:36861429 Surfaces of crystalline gypsum Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) The affinity of selected oligopeptides and synthesized copolymers to bind to the surface of gypsum and C-S-H particles was assessed in batch assays based on total organic carbon (TOC) analysis. For the selection phase, the following procedure was used: 10 µL of the original phage library solution was mixed with 990 µL saturated gypsum solution (to prevent the dissolution of the gypsum particles during the experiment) in standard Eppendorf vials. To this dilution, solid gypsum powder was added at a ratio of 0.1% (w/v) and the resulting suspension was incubated at room temperature for 30 min while shaking at 800 rpm. Afterward, the solid particles carrying the strongly adhering phages were separated by centrifugation for 30 s at 8000 rpm, while the non- and weakly bound phages remained in the supernatant, which was removed. The remaining solid material was washed ten times with 1 mL saturated gypsum solution by vortexing for 15 s and subsequent centrifugation, in order to further separate weakly from strongly binding phages. During the washing sequence, the Eppendorf vial was replaced three times to eliminate any potential influence of phages sticking to the polypropylene surface of the vials (i.e., to increase the selection pressure and remove peptide sequences with enhanced affinity for the vial surfaces prior to analysis). To isolate the strongly bound phages in the last step, the washed gypsum particles were dissolved in a mixture of 1 mL Tris/HCl and 100 µL Tris-buffered saline. Subsequently, the released phages were amplified in a 20 mL lysogeny broth (LB) medium containing 400 µL Escherichia Coli solution (strain ER2738, OD600 ≈ 0.5). After 4-5 hours of vigorous shaking at 37 °C, the phages were reprocessed according to the protocol of the supplier to prepare the selected library for another panning round. Based on next-generation sequencing of phages enriched during the screening process, a triplet of amino acids, DYH, is identified as the main driver for adsorption on the mineral substrate. Furthermore, oligopeptides containing this motif prove to exert their influence in a strictly selective manner during the hydration of cement, where the sulfate reaction (initial setting) is strongly retarded while the silicate reaction (final hardening) remains unaffected. In the final step, these desired additive characteristics are successfully translated from the level of peptides to that of scalable synthetic copolymers. 3579 DYHDPSLPTLRK(76.0%)[0.0321±0.0062] TAKYLPMRPGPL(7.9%)[NT] QVNGLGERSQQM(4.3%)[NT] RDYHPRDHTATW(2.6%)[NT] GNNPLHVHHDKR(1.2%)[NT] SPLRAVAFSGAQ(0.6%)[NT] INIVPGPEKPVG(0.5%)[NT] QGYKQEYTRWGE(0.1%)[NT] 8 Phage display (common panning) 2 PMID:36861429 Surfaces of crystalline gypsum Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) The affinity of selected oligopeptides and synthesized copolymers to bind to the surface of gypsum and C-S-H particles was assessed in batch assays based on total organic carbon (TOC) analysis. For the selection phase, the following procedure was used: 10 µL of the original phage library solution was mixed with 990 µL saturated gypsum solution (to prevent the dissolution of the gypsum particles during the experiment) in standard Eppendorf vials. To this dilution, solid gypsum powder was added at a ratio of 0.1% (w/v) and the resulting suspension was incubated at room temperature for 30 min while shaking at 800 rpm. Afterward, the solid particles carrying the strongly adhering phages were separated by centrifugation for 30 s at 8000 rpm, while the non- and weakly bound phages remained in the supernatant, which was removed. The remaining solid material was washed ten times with 1 mL saturated gypsum solution by vortexing for 15 s and subsequent centrifugation, in order to further separate weakly from strongly binding phages. During the washing sequence, the Eppendorf vial was replaced three times to eliminate any potential influence of phages sticking to the polypropylene surface of the vials (i.e., to increase the selection pressure and remove peptide sequences with enhanced affinity for the vial surfaces prior to analysis). To isolate the strongly bound phages in the last step, the washed gypsum particles were dissolved in a mixture of 1 mL Tris/HCl and 100 µL Tris-buffered saline. Subsequently, the released phages were amplified in a 20 mL lysogeny broth (LB) medium containing 400 µL Escherichia Coli solution (strain ER2738, OD600 ≈ 0.5). After 4-5 hours of vigorous shaking at 37 °C, the phages were reprocessed according to the protocol of the supplier to prepare the selected library for another panning round. Based on next-generation sequencing of phages enriched during the screening process, a triplet of amino acids, DYH, is identified as the main driver for adsorption on the mineral substrate. Furthermore, oligopeptides containing this motif prove to exert their influence in a strictly selective manner during the hydration of cement, where the sulfate reaction (initial setting) is strongly retarded while the silicate reaction (final hardening) remains unaffected. In the final step, these desired additive characteristics are successfully translated from the level of peptides to that of scalable synthetic copolymers. 3580 DYHDPSLPTLRK(86.3%)[0.0321±0.0062] TAKYLPMRPGPL(7.8%)[NT] QVNGLGERSQQM(0.9%)[NT] RDYHPRDHTATW(0.7%)[NT] GNNPLHVHHDKR(0.4%)[NT] SPLRAVAFSGAQ(0.1%)[NT] INIVPGPEKPVG(0.1%)[NT] QGYKQEYTRWGE(0.1%)[NT] 8 Phage display (common panning) 3 PMID:36861429 Surfaces of crystalline gypsum Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) The affinity of selected oligopeptides and synthesized copolymers to bind to the surface of gypsum and C-S-H particles was assessed in batch assays based on total organic carbon (TOC) analysis. For the selection phase, the following procedure was used: 10 µL of the original phage library solution was mixed with 990 µL saturated gypsum solution (to prevent the dissolution of the gypsum particles during the experiment) in standard Eppendorf vials. To this dilution, solid gypsum powder was added at a ratio of 0.1% (w/v) and the resulting suspension was incubated at room temperature for 30 min while shaking at 800 rpm. Afterward, the solid particles carrying the strongly adhering phages were separated by centrifugation for 30 s at 8000 rpm, while the non- and weakly bound phages remained in the supernatant, which was removed. The remaining solid material was washed ten times with 1 mL saturated gypsum solution by vortexing for 15 s and subsequent centrifugation, in order to further separate weakly from strongly binding phages. During the washing sequence, the Eppendorf vial was replaced three times to eliminate any potential influence of phages sticking to the polypropylene surface of the vials (i.e., to increase the selection pressure and remove peptide sequences with enhanced affinity for the vial surfaces prior to analysis). To isolate the strongly bound phages in the last step, the washed gypsum particles were dissolved in a mixture of 1 mL Tris/HCl and 100 µL Tris-buffered saline. Subsequently, the released phages were amplified in a 20 mL lysogeny broth (LB) medium containing 400 µL Escherichia Coli solution (strain ER2738, OD600 ≈ 0.5). After 4-5 hours of vigorous shaking at 37 °C, the phages were reprocessed according to the protocol of the supplier to prepare the selected library for another panning round. Based on next-generation sequencing of phages enriched during the screening process, a triplet of amino acids, DYH, is identified as the main driver for adsorption on the mineral substrate. Furthermore, oligopeptides containing this motif prove to exert their influence in a strictly selective manner during the hydration of cement, where the sulfate reaction (initial setting) is strongly retarded while the silicate reaction (final hardening) remains unaffected. In the final step, these desired additive characteristics are successfully translated from the level of peptides to that of scalable synthetic copolymers. 3581 VIELVILIDD APELLHLIDE HKELSQLIWF PKELNRLIFG YGELGYLIHG EHELTLLILF YMELFILIAI SIELRHLIYE GDELHKLILY FWELNILIVY 10 Phage display (common panning) 3 PMID:35580187 Amyloid β peptide (Aβ) (M1-40) monomer, Aβ40 monomer Not determined. Not determined. Not determined. pIT2 X2ELX2LIX2 phage display library 3582 DQELEGLIHP SLELFPLIDD DAELNKLINP AYELPYLIIP IWELNILIEL AIELYALIFK DYELPNLIPP EFELGPLIEA GNELMQLIMM YYELDDLIMP 10 Phage display (common panning) 3 PMID:35580187 Amyloid β peptide (Aβ) (M1-40) fibril, Aβ40 fibril Not determined. Not determined. Not determined. pIT2 X2ELX2LIX2 phage display library 3583 TAELRNLIWA THELPILILS MAELHALIGY MRELVELIHV NEELEHLINE GPELYALIIF HNELPDLIQL HSELELLIEY VMELANLIGT HFELSRLINY 10 Phage display (common panning) 3 PMID:35580187 Amyloid β peptide (Aβ) (M1-42) monomer, Aβ42 monomer Not determined. Not determined. Not determined. pIT2 X2ELX2LIX2 phage display library 3584 WTELTVLIVW PTELQILIHW TPELAILISY PPELQNLIHF FLELDVLIHA YYELTELIYV RQELGFLILF KNELYWLIVE DSELKYLIYW DDELFMLIRM 10 Phage display (common panning) 3 PMID:35580187 Amyloid β peptide (Aβ) (M1-42) fibril, Aβ42 fibril Not determined. Not determined. Not determined. pIT2 X2ELX2LIX2 phage display library 3585 GGGVNIGLEYGGG GGGLFVMTRMGGG GGGHHYTVFMGGG GGGILALFFVGGG GGGEDHREMDGGG GGGHPRSTAVGGG GGGIMGYPLNGGG GGGFGVHEWVGGG GGGPTDIWAWGGG GGGPIHESEHGGG 10 Phage display (common panning) 3 PMID:35580187 Amyloid β peptide (Aβ) (M1-40) monomer, Aβ40 monomer STAT3 Y705 specific antibody Not determined. Not determined. pIT2 GGGX7GGG phage display library 3586 GGGIRQDAQAGGG GGGRHRKPFEGGG GGGGLDTRHDGGG GGGTLGKMHHGGG GGGKNMQMWVGGG GGGDYQPQGIGGG GGGWPVGHATGGG GGGMKQGPVYGGG GGGFKRSWIFGGG GGGHITHNETGGG 10 Phage display (common panning) 3 PMID:35580187 Amyloid β peptide (Aβ) (M1-40) fibril, Aβ40 fibril Not determined. Not determined. Not determined. pIT2 GGGX7GGG phage display library 3587 GGGQGKSVPAGGG GGGYLTIRLMGGG GGGASNTYFSGGG GGGLIWGFKTGGG GGGPDPLDFDGGG GGGDLVSFYYGGG GGGSWMALLVGGG GGGSSWRGTTGGG GGGHHQMTKSGGG GGGPWTVPVDGGG 10 Phage display (common panning) 3 PMID:35580187 Amyloid β peptide (Aβ) (M1-42) monomer, Aβ42 monomer Not determined. Not determined. Not determined. pIT2 GGGX7GGG phage display library We identified an SXkmer with loop–insertion YLTIRLM as an inhibitor of the secondary nucleation of Aβ42 and binding analyses using surface plasmon resonance technology, F€orster resonance energy transfer, and microfluidics diffusional sizing imply an interaction with intermediate oligomeric species. A linear peptide with the YLTIRLM sequence was found inhibitory but at a lower potency than the more constrained SXkmer loop. We identified an SXkmer with side-patch VI-WI-DD as an inhibitor of Aβ40 aggregation. Remarkably, our data imply that SXkmer-YLTIRLM blocks secondary nucleation through an interaction with oligomeric intermediates in solution or at the fibril surface, which is a unique inhibitory mechanism for a library-derived inhibitor. 3588 GGGEGVNEFFGGG GGGIRWTVMMGGG GGGGYRWWWVGGG GGGDRSNSPEGGG GGGRAHDASIGGG GGGVHTKAAAGGG GGGDQWIEHVGGG GGGNEMFVVWGGG GGGDRQWYPAGGG GGGVHKFGHIGGG 10 Phage display (common panning) 3 PMID:35580187 Amyloid β peptide (Aβ) (M1-42) fibril, Aβ42 fibril Not determined. Not determined. Not determined. pIT2 GGGX7GGG phage display library 3589 CPTTMWRYC(13)[1.4229±0.2161] CSTPMWKYC(5)[NT] CEKMVATHC(2)[NT] CTPRSANYC(2)[NT] CDGANARMC(1)[NT] CDGAPDAAC(1)[NT] CDQSVPHSC(1)[NT] CFGYYGPPC(1)[NT] CGSLDWPHC(1)[NT] CGTTEWRYC(1)[NT] CHNTYHRLC(1)[NT] CIVEASVHC(1)[NT] CMSWSNYFC(1)[NT] CNSSKLHMC(1)[NT] CPFWPSGHC(1)[NT] CPPMGOGNC(1)[NT] CPTTMWKHC(1)[NT] CQASRPALC(1)[NT] CQSCYCNHC(1)[NT] CQSYEPLRC(1)[NT] CRSTPWPSC(1)[NT] CSPHLNTNC(1)[NT] CSPWSYLFC(1)[NT] CSRSMDSTC(1)[NT] CSTGNNFAC(1)[NT] CSTLHQKLC(1)[NT] CTDKASSSC(1)[NT] CTKPNAPMC(1)[NT] CTNSGTSGC(1)[NT] CTSPMWRYC(1)[NT] CTTDVTGRC(1)[NT] GINNLPKSC(1)[NT] 32 Phage display (competitive panning) 2-4 PMID:35240465 Caspase-3, CASP-3 Not determined. Not determined. Not determined. Ph.D.-C7C phage display library (CX7C) ELISA First, the streptavidin-coated 96-well plates were pre-washed three times with 200 μL of 0.1 M PBS before protein immobilization, and the biotinylated caspase-3 was added to the functionalized streptavidin-coated plate and stirred at 110 rpm for 1 h. Subsequently, the unbound proteins were removed and then added blocking solution (5% BSA in NaHCO3, pH 8.6) at 4 ℃ for 1.5 h. Then, the wells were washed six times with PBST buffer, and each phage were added at a concentration of 1012 PFU/mL, followed by stirring at 110 rpm at room temperature for 1 h. After washing six times with same buffer, horseradish peroxidase (HRP)-conjugated anti-M13 monoclonal antibody (1:2500 dilution in blocking buffer) was added and incubated for 1 h. The remaining solution was removed, and the plate was washed again with the same buffer. The ABTS and HRP substrate were added, and absorbance was measured at 405 nm (Multiskan, Thermo Scientific, Waltham, MA, USA). Absorbance at 405 nm was reproduced from Figure 1A. In the first round of biopanning, the biotinylated caspase-3 was separately pre-reacted with the Ph.D.-C7C random phage libraries (∼e11 PFU/mL) at 100 rpm for 1 h at room temperature. After mild agitation of the phage-protein complexes, 100 μL of the complexes was introduced to the streptavidin-coated 96-well plates (Thermo Scientific) and allowed to react at 110 rpm for 10 min to facilitate specific binding between avidin and biotin. Then, 1 μL of biotin (0.1 mM) was added to competitively remove the phages which is non-specifically binding to the streptavidin-coated plate as a blocking agent and incubated for 5 min, followed by repeated washing 10 times using PBST buffer containing 0.1 M PBS with 0.1% Tween 20 for removing unbound phages and residual biotin. Finally, the bound phages were eluted in 0.2 M glycine-HCl (pH 2.2) with 1 mg/mL BSA solution. The eluent was neutralized with 15 μL of Tris-HCl (pH 9.0) to prevent deactivation or destruction of the desired phages. The eluted phages were amplified using Escherichia coli ER2738, and the amplified phage solution was quantified by phage titration for subsequent rounds of biopanning. During this biopanning, negative biopanning against streptavidin-coated plate was performed with the amplified phages in the beginning of the second round, and Tween 20 concentration of PBST was gradually increased (0.1–0.5%) for removing non-specifically binding phages. We identified potential affinity peptide-displayed phage clones with the sequence CPTTMWRYC. After characterization of its binding affinity using enzyme-linked immunosorbent assay, whole phage particles were covalently attached to a gold surface using coupling chemistry (MUA-EDC/NHS). The developed phage sensor was characterized by X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), scanning electron microscopy (SEM), electrochemical analysis using cyclic voltammetry (CV), and square wave voltammetry (SWV). Under optimal conditions, the affinity peptide-displayed phage sensor showed a good binding affinity (Kd = 0.13 ± 0.56 μM) and limit of detection (0.39 μM) for caspase-3 detection. Furthermore, developed phage sensor could be monitored the response of apoptotic HeLa cells by detecting caspase-3 activity. This work should stimulate the development of efficient alternative caspase-3 detection methods for the diagnosis and prognosis of apoptosis-related diseases. 3590 NWYLPWLGTNDW[++++] TWDLPWLLEKPF[++++] KMLPTMPRVLAG[++] DAAPTLPKGGVG[++] QIDTGYGLVSVS[++] GSKTGYLSETVR[++] ASKNAHLFLSSL[+] QQQYGTYVPTFG[+] 8 Phage display (subtractive panning) 3 PMID:37046671 M2 macrophages Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) Flow Cytometry Binding of the phage clones to M2 macrophages was analyzed by flow cytometry. +, binding < 25%; ++, binding < 50%; ++++, binding > 80–100%. For the NW peptide (NWYLPWLGTNDW) blocking strategy, the M2 macrophages were incubated with 200 μg of the NW peptide for 1 h at RT prior to biopanning as described above. Three rounds of biopanning were performed on blocked M2 macrophages. Similarly, the library was pre-incubated with peripheral blood mononuclear cells (PBMCs) prior to affinity selection on blocked M2 macrophages. 3591 NWYLPWLGTNDW[++++] QWELPWLMQPPL[++++] TWALPWLLEKPF[++++] SPILWLNAPPWA[++++] WHDLWSSNWDTV[++++] GENLMSVGLLRT[++] 6 Phage display (subtractive panning) 3 PMID:37046671 M2 macrophages Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) Flow Cytometry Binding of the phage clones to M2 macrophages was analyzed by flow cytometry. +, binding < 25%; ++, binding < 50%; ++++, binding > 80–100%. The 12-mer peptide phage library (Ph.D.™-12) was purchased from New England BioLabs (Ipswich, MA, USA). The phage library was amplified and titered according to the manufacturer’s instructions. The M1 and M2 macrophages used for biopanning were grown in T25 flasks until they reached 80–90% confluency on the day of the experiment. Prior to biopanning, the cells were washed in PBS buffer supplemented with 5% bovine serum albumin (BSA) (washing buffer) to remove dead cells. Thereafter, the phage library (1011 transduction units, TU), diluted in 3 mL washing buffer, was added to the cells and incubated for 1 h at room temperature with gentle agitation. Subsequently, the supernatant was removed and 10 mL washing buffer was added to the cells. The cells were then harvested by gentle scraping and transferred to a 15 mL Falcon tube. The cells were pelleted by centrifugation at 300× g for 3 min and then washed 10 times with the washing buffer to remove unbound phages. Cell-binding phages were eluted in 200 μL elution buffer (0.1 M glycine-HCl, pH2.2, 0.1% BSA) for 10 min at RT with constant rotation followed by centrifugation for 5 min at 12,000× g. The supernatant containing the eluted phages was collected and neutralized with 28 μL neutralization buffer (Tris-HCl, pH 9.2). The eluted phages were amplified in the E. coli ER2738 strain, titered and then used in subsequent rounds of biopanning. In addition, the biopanning was performed with subtraction steps, in which the phage library was pre-incubated with peripheral blood mononuclear cells and/or M1 macrophages prior to affinity selection on M2 macrophages. This pre-incubation step was carried out from round 1. To explore the therapeutic potential of the selected peptides, the M13 phage-displayed peptides were conjugated to the photosensitizer IR700, which has been used for cancer photoimmunotherapy. The phage displaying a dominant peptide (SPILWLNAPPWA) killed both M1 and M2 macrophages, while those displaying the M2-specific peptides killed M2 macrophages only upon near-infrared light exposure. A significant fraction of the M2 macrophages were also killed with the untargeted M13 phage-IR700 conjugates. Hence, M2 macrophages can also be selectively targeted by the wild type M13 phage, which displayed a significant tropism to these cells. The benefits of this photoimmunotherapy include an automatic self-targeting ability of the wild type M13 phage, and the option of genetic manipulation of the phage genome to include tumor targeting peptides, allowing the killing of both M2 macrophages and cancer cells. 3592 LTPHKHHKHLHA(11)[0.4363±0.0133] STPHKHHKHLHA(5)[0.378±0.012] FFADVTKHSQKT(2)[0.2969±0.0243] HPASGWHMRHHR(1)[0.2005±0.0083] 4 Phage display (competitive panning) 4 PMID:35700851 Immunomodulatory protein Ling Zhi-8, LZ-8 mesenchymal stem cells (MSCs) Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA Absorption was measured at 450 nm. Data shown were reproduced from Figure 1A. A specific elution was performed for the fourth round of biopanning, in which the rLZ-8-bound phages were eluted with rLZ-8 polyclonal antibody after washing with TBS-T 0.5% (v/v). rLZ-8 is short for recombinant Ganoderma lucidum immunomodulatory protein-8. The binding mode between of recombinant Ganoderma lucidum immunomodulatory protein-8 (rLZ-8) and the peptide ligand (LTPHKHHKHLHA) was simulated and revealed by molecular docking. Standard addition and repetitive testing were carried out to evaluate the accuracy, reproducibility and feasibility of the developed ELISA detection method. The method based on this peptide ligand was then successfully applied in the quantitative determination of rLZ-8 concentrations in fermentation broth. In summary, the peptide–antigen–antibody sandwich ELISA method developed here could be conveniently applied in the detection of rLZ-8 during fermentation and might provide new insights for the detection of other specific proteins. 3593 TSATKFMMNLSP(24)[0.5012±0.1015] 1 Phage display (subtractive panning) 6 PMID:35323423 Klebsiella pneumoniae KCTC 2208 cell Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA The microtiter plates were coated with bacterial cells (1 × 107 colony-forming unit (CFU)/well) overnight at 4 °C and incubated again with 3% BSA in TBS for 1 h at room temperature to block non-specific binding. After washing three times with TBST, one hundred microliters of amplified phages (1 × 109 plaque-forming unit) were added and incubated for 1 h. The wells were washed three times with TBST; subsequently, horseradish peroxidase (HRP)-conjugated anti-M13 monoclonal antibody (1:5000, GE Healthcare, Chicago, IL, USA) was added and incubated for 1 h. The wells were rewashed with TBST, and 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) peroxidase substrate (Sigma-Aldrich, St. Louis, MO, USA) was added to the wells to detect phage binding. The color development at 415 nm was recorded using a microplate reader (Synergy, BioTeK, Santa Clara, CA, USA). Data shown were reproduced from Figure 1. To ensure specificity, the phage library was depleted of clones binding to bovine serum albumin (BSA)-coated wells before selecting Klebsiella pneumoniae binding clones. The biopanning-derived peptide TSATKFMMNLSP, KP peptide, displayed a high selectivity for the K. pneumoniae with low cross-reactivity to related Gram-negative bacteria. The specific interaction between KP peptide and K. pneumoniae lipopolysaccharide resulted in the peptide’s selectivity against K. pneumoniae. Quantitative analysis of this interaction by enzyme-linked immunosorbent assay revealed that the KP peptide possessed higher specificity and sensitivity toward K. pneumoniae than commercially available anti-Klebsiella spp. antibodies and could detect K. pneumoniae at a detection limit of e4 CFU/mL. These results suggest that KP peptide can be a promising alternative to antibodies in developing a biosensor system for K. pneumoniae detection. 3594 KWHWKDKNALRM(5/15) GPVNKSSTILRM(3/15) GSLRPGTTNALV(2/15) GLHTSATNLYLH(2/15) AHGNAALVARLK(1/15) FGDLTRGQQRGP(1/15) QGTVARLPIFWP(1/15) 7 Phage display (common panning) 4 PMID:36979401 Pseudomonas aeruginosa ATCC 27853 Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA The color development in the wells was recorded by FLUOstar Omega microplate reader (BMG LabTech) at the wavelength of 405 nm. Data were not shown. Peptides that bind to P. aeruginosa were selected via biopanning of a 12-mer random phage-displayed peptide library against the bacterial whole cells according to the protocol as described by the manufacturer (New England Biolabs, Ipswich, MA, USA) [28]. For the first round of biopanning, P. aeruginosa ATCC 27853 resuspended in 2 mL of PBS (pH 7.4) was added with 10 µL of the phage library diluted in 100 µL of TBST, followed by incubation at room temperature for 60 min with gentle agitation. Unbound phages were removed by a series of washing with TBST followed by centrifugation. Bound phages were eluted with 200 µL of elution buffer [0.2 M Glycine-HCl (pH 2.2), 1 mg/mL BSA] for 10 min at room temperature, followed by neutralization of the eluted phages with 30 µL of 1 M Tris-HCl (pH 9.1). A titer of the eluted phages was determined by plating an aliquot of the phages on LB IPTG/Xgal agar, while the rest of the phages were amplified in Escherichia coli ER2738. Amplified phages were purified with polyethylene-glycol precipitation, followed by resuspension of the phage pellet in TBS. The titer of the amplified phages was determined before proceeding to the subsequent round of biopanning. A total of four rounds of biopanning were carried out to enrich the clones of phage-displayed peptides binding to P. aeruginosa, with the titers of input phages (phages in TBS after amplification) and output phages (phages in elution buffer) being determined each round. After the final round of biopanning, individual eluted phages on the titer plate were randomly selected for amplification in E. coli ER2738 to be used in phage-ELISA and phage genomic DNA extraction. The affinity-selected peptide (KWHWKDKNALRM) with the highest selection frequency was modified to PAM-5 (KWKWRPLKRKLVLRM) with enhanced antibacterial features by using an online peptide database. Using in vitro microbroth dilution assay, PAM-5 was shown to be active against a panel of Gramnegative bacteria and selected Gram-positive bacteria. Interestingly, the peptide was stable in human plasma by exhibiting a similar bactericidal effect via ex vivo assay. Scanning electron microscopy and SYTOX Green uptake assay revealed that PAM-5 was able to cause membrane disruption and permeabilization of the bacteria. Additionally, the peptide was also able to bind to bacterial DNA as demonstrated by gel retardation assay. In the time-kill assay, PAM-5 was shown to kill the bacteria rapidly in 10 min. More importantly, PAM-5 was non-cytotoxic to Vero cells and non-haemolytic to human erythrocytes at all concentrations tested for the antibacterial assays. 3595 ACEGLYAHWC[0.2756±0.0275] 1 Phage display (common panning) 3 PMID:34259504 Anti-mycophenolic acid (MPA) antibody fragment (Fab), anti-MPA Fab Mycophenolic acid, MPA Not determined. Not determined. Ph.D.-C7C phage display library (CX7C) ELISA Absorbance at 405 nm was measured in a Varioskan plate reader (Thermo Scientific). Data were reproduced from Figure 1A. A commercial phage-displayed peptide library was used to select cyclic peptides that bind to the anti-MPA. The selection rounds were carried out with an automatic magnetic bead processor (KingFisher Thermo Fisher Scientific). See the Supporting Information for antibody coupling to magnetic beads. Briefly, the phage-displayed peptide library (∼2.0 × 1011 phages) was incubated for 2 h with the anti-MPA conjugated beads (50 μg) in a total volume of 505 μL of PBST [PBS, pH 7.4 with 0.05% (v/v) Tween-20]. The beads were subsequently washed twice with PBST for 30 s, and then the bound phages were eluted with 100 μL of 0.1 M triethylamine (pH 11.2) for 30 min. The resulting solution containing the eluted phages was immediately neutralized with 70 μL of 1 mol L–1 Tris-HCl (pH 6.8). Amplification of the eluted phages was carried out by adding 70 μL of the eluate to a 40 mL early-log phase ER2738 culture in LB and incubating at +37 °C for 4.5 h. The cells were harvested by centrifugation (10 min, 12,000g, +4 °C), and the supernatant was collected. The amplified phages were precipitated overnight at +4 °C after adding to the supernatant 1/6 volume of 20% poly(ethylene glycol) (PEG)/2.5 mol L–1 NaCl. Then, the precipitated phages were collected by centrifugation (15 min, 12,000g, +4 °C) and resuspended in 3 mL of PBS. The precipitation was repeated with 20% PEG/2.5 mol L–1 NaCl on ice for 1 h, followed by centrifugation (10 min, 12,000g, +4 °C). Finally, the pellet containing the phages was resuspended in 500 μL of PBS. The amplified phage solution was utilized for the consequent selection round. After identifying the best mycophenolic acid (MPA) mimetic (ACEGLYAHWC with a disulfide constrained loop), several immunoassay approaches were tested, and a recombinant fusion protein containing the peptide sequence with a bioluminescent enzyme, NanoLuc, was developed. The recombinant fusion enabled its direct use as the tracer in competitive immunoassays without the need for secondary antibodies or further labeling. A bioluminescent sensor, using streptavidin-coupled magnetic beads for the immobilization of the biotinylated Fab antibody, enabled the detection of MPA with a detection limit of 0.26 ng mL−1 and an IC50 of 2.9 ± 0.5 ng mL−1. The biosensor showed good selectivity toward MPA and was applied to the analysis of the immunosuppressive drug in clinical samples, of both healthy and MPA-treated patients, followed by validation by liquid chromatography coupled to diode array detection. 3596 AAHRVGGFNYHM(7/20)[1097.7413±82.1182] SVPLNSWSIFPR(5/20)[1526.2958±108.2934] STVYHTTPYHNR(3/20)[NT] 3 Phage display (common panning) PMID:37098917 Staphylococcus aureus, S.aureus Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA An ELISA plate was coated with an S. aureus suspension using the methods described in “Two panning methods for phage selection,” above. Serial dilutions of the phage in 100 μL of TBST were prepared, added to each well, and then incubated at RT for 1 h with agitation. The plate was washed six times with TBST; then, 200 μL of the diluted HRP-conjugated anti-M13 monoclonal antibody (GE Healthcare) was added to each well and incubated at RT for 1 h with agitation. The HRP substrate solution was prepared according to the manufacturer’s instructions. Finally, 200 μL of substrate solution was added to each well and incubated for 60 min at room temperature (RT) with gentle agitation, and the plate was read at 405 to 415 nm. Staphylococcus aureus cells were cultured in Luria-Bertani (LB) medium until the optical density at 600 nm (OD600) reached 0.6 to 0.8. The cells were then collected by centrifugation, washed with phosphate-buffered saline (PBS), and mixed with ethanol before being dried on a 96-well plate (100 μL/well) at room temperature (RT) overnight. For the first panning round, the cells fixed on the 96-well plate were exposed to e11 PFU/mL of phage library in TBST buffer (Tris-buffered saline with 0.05% Tween 20, pH 7.5) (100 μL/well). Unbound phages were eliminated by washing five times with TBST, and the bound phages were eluted with 100 μL elution buffer (0.2 M Glycine-HCl) for 20 min at room temperature. The eluted phage was neutralized with 1 M Tris-HCl (pH 9.1) and amplified in E. coli ER2738, followed by purification with polyethylene glycol precipitation. A phage clone displaying a peptide capable of specific binding to a whole S. aureus cell was selected from a 12-mer phage peptide library. The peptide sequence was SVPLNSWSIFPR. The selected phage’s ability to bind specifically with S. aureus was confirmed using an enzyme-linked immunosorbent assay, and the chosen peptide was then synthesized. The results showed that the synthesized peptides displayed high affinity with S. aureus but low binding ability with other strains, including Gram-negative and Gram-positive bacteria such as Salmonella sp., Shigella spp., Escherichia coli, and Corynebacterium glutamicum. In addition, yeast vacuoles were used as a drug carrier by encapsulating daptomycin, a lipopeptide antibiotic used to treat Gram-positive bacterial infections. The expression of specific peptides at the encapsulated vacuole membrane created an efficient system that can specifically recognize and kill S. aureus bacteria.  3597 VGQFGTSQMILP(6/20)[1168.5683±108.2934] SWPTFTVLKNHA(3/20)[NT] NFTLQAHPHKYP(3/20)[NT] 3 Phage display (common panning) PMID:37098917 Staphylococcus aureus, S.aureus Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA An ELISA plate was coated with an S. aureus suspension using the methods described in “Two panning methods for phage selection,” above. Serial dilutions of the phage in 100 μL of TBST were prepared, added to each well, and then incubated at RT for 1 h with agitation. The plate was washed six times with TBST; then, 200 μL of the diluted HRP-conjugated anti-M13 monoclonal antibody (GE Healthcare) was added to each well and incubated at RT for 1 h with agitation. The HRP substrate solution was prepared according to the manufacturer’s instructions. Finally, 200 μL of substrate solution was added to each well and incubated for 60 min at room temperature (RT) with gentle agitation, and the plate was read at 405 to 415 nm. The e11 PFU/mL of Ph.D.-12 phages was exposed to S. aureus cells (OD600, 0.5) in PBS for 60 min at RT with gentle agitation. The bacteria-phage mixture was centrifuged for 5 min at 16,000 × g, and then the unbound phages were removed by washing 10 times (centrifugation at 16,000 × g for 5 min with 1 mL TBST). The pellet that contained the bound phages was eluted with 250 μL of 0.2 M glycine-HCl (pH 2.2) with slight shaking at RT for 20 min and was then sonicated for 10 min with 50% amplitude, 30 s on and 30 s off. Subsequently, the solutions were neutralized with 25 μL of 1 M Tris-HCl (pH 9). The eluate (10 μL) would be used for phage titering, and 500 μL was used to infect E. coli ER2738 for amplification. A phage clone displaying a peptide capable of specific binding to a whole S. aureus cell was selected from a 12-mer phage peptide library. The peptide sequence was SVPLNSWSIFPR. The selected phage’s ability to bind specifically with S. aureus was confirmed using an enzyme-linked immunosorbent assay, and the chosen peptide was then synthesized. The results showed that the synthesized peptides displayed high affinity with S. aureus but low binding ability with other strains, including Gram-negative and Gram-positive bacteria such as Salmonella sp., Shigella spp., Escherichia coli, and Corynebacterium glutamicum. In addition, yeast vacuoles were used as a drug carrier by encapsulating daptomycin, a lipopeptide antibiotic used to treat Gram-positive bacterial infections. The expression of specific peptides at the encapsulated vacuole membrane created an efficient system that can specifically recognize and kill S. aureus bacteria.  3598 LPSYNLHPHVPP(3/12) IPLLNPGSMQLS(4/12) 2 Phage display (subtractive panning) 2 PMID:36303134 Human lens capsules from patients with exfoliation syndrome (XFS) Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) Human lens capsules collected from patients without exfoliation syndrome (XFS) were used for subtractive screening. peptides differentiate between exfoliative and non-affected regions of the human lens capsule 3599 LPSYNLHPHVPP(11/52) IPLLNPGSMQLS(37/52) 2 Phage display (subtractive panning) 3 PMID:36303134 Human lens capsules from patients with exfoliation syndrome (XFS) Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) Human lens capsules collected from patients without exfoliation syndrome (XFS) were used for subtractive screening. peptides differentiate between exfoliative and non-affected regions of the human lens capsule 3600 HDYLYYTFTGNP(4)[1.1555±0.0301] TKFSPPSFWYLH(2)[1.3806±0.0245] GPFWPTQGAHLR(2)[NT] WTRKYMPYGPTP(2)[NT] GPGLMLRPAFSN(1)[NT] ETDYHWMNYLFS(1)[NT] HAHVIYSPHLPP(1)[NT] AVTLTQLSPQIH(1)[NT] 8 Phage display (common panning) 4 PMID:33340637 Aminopeptidase Ey Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA The absorbance was measured at 490 nm after using OPD color reagent for 10 min. Biopanning of the phage was performed using the Ph.D™-12 Phage Display Peptide Library Kit (New England Biolabs, Beverly, MA, USA) according to the manufacturer's instructions with minor modifications. In brief, ELISA plates were coated with rgAPN protein (2 μg/ml) per well overnight at 4 °C. The original phage library (1.5 × 1011 pfu/well) was incubated. Phages in 1st round biopanning were obtained by eluting (0.2 mol/L Glycine-HCl), and then neutralized (1 mol/L Tris-HCl). The treatments were replicated 4 times. Titers of phages for each round were determined on LB/IPTG/Xgal (Low LB + 0.1% [v/v] IPTG/Xgal) plates. High-affinity ligands of pET-gAPN protein, H (HDYLYYTFTGNP) and T (TKFSPPSFWYLH), screened by the phage display peptide library, reduced IBV infection in vivo and in vitro via blocking the key antigenic motifs, and might provide a novel countermeasure for IB prevention or treatment. In addition, the results indirectly demonstrated that gAPN acts as a receptor for IBV.