Phage-displayed Peptide Research Articles

Phage Display in the Quest for New Selective Recognition Elements for Biosensors.

Phages are bacterial viruses that have gained a significant role in biotechnology owing to their widely studied biology and many advantageous characteristics. Perhaps the best-known application of phages is phage display that refers to the expression of foreign peptides or proteins outside the phage virion as a fusion with one of the phage coat proteins. In 2018, one half of the Nobel prize in chemistry was awarded jointly to George P. Smith and Sir Gregory P. Winter “for the phage display of peptides and antibodies.” The outstanding technology has evolved and developed considerably since its first description in 1985, and today phage display is commonly used in a wide variety of disciplines, including drug discovery, enzyme optimization, biomolecular interaction studies, as well as biosensor development. A cornerstone of all biosensors, regardless of the sensor platform or transduction scheme used, is a sensitive and selective bioreceptor, or a recognition element, that can provide specific binding to the target analyte. Many environmentally or pharmacologically interesting target analytes might not have naturally appropriate binding partners for biosensor development, but phage display can facilitate the production of novel receptors beyond known biomolecular interactions, or against toxic or nonimmunogenic targets, making the technology a valuable tool in the quest of new recognition elements for biosensor development.

Functional mimetic peptide discovery isolated by phage display interacts selectively to fibronectin domain and inhibits gelatinase.

Matrix metalloproteinases (MMPs) play critical roles in a multiple number of autoimmunity diseases progression and metastasis of solid tumor. Gelatinases including MMP-2 and MMP-9 are extremely overexpressed in multiple pathological processes. MMP-9 and MMP-2 breakdown the extracellular matrix component gelatin very efficaciously. Therefore, designing and expansion of MMPs inhibitors can be an engrossing plan for therapeutic intermediacy. Anyway, a wide range of MMPs inhibitors face failure in several clinical trials. Due to sequence and structural conservation across the various MMPs, achieving specific and selective inhibitors is very demanding. In the current study, a phage-displayed peptide library was screened using active human recombinant MMP-9 protein and evaluated by enzyme-linked immunosorbent assay. Here, we isolate novel peptide sequence from phage display peptide libraries that can be a specific gelatinase inhibitor. Interestingly, in silico molecular docking showed strong interactions between the peptide three-dimensional models and some important residues of the MMP-9 and MMP-2 proteins at the fibronectin domain. A consensus peptide sequence was then synthesized (named as RSH-12) to evaluate its inhibitory potency by in vitro assays. Zymography assay was employed to evaluate the effect of RSH-12 on gelatinolysis activity of MMP-2 and MMP-9 secretion from the HT1080 cells using different concentrations of RSH-12 and inhibiting MMP-9- and MMP-2-driven gelatin proteolysis, measured by fluorescein isothiocyanate-gelatin degradation assay and HT1080 cellinvasion assay on Matrigel (gelatinous protein mixture). The negative control peptide (CP) with the irrelevant sequence and no MMP inhibition properties and the positive control compound (GM6001) as a potent inhibitor of MMPs were used to assess the selectivity and specificity of gelatinases inhibition by RSH-12. Therefore, RSH-12 decreased the gelatin degradation by specifically preventing gelatin binding to MMP-9 and MMP-2. Selective gelatinase inhibitors may prove the usefulness of the new peptide discovered in tumor targeting and anticancer and anti-inflammation therapies.

Alternaria B Cell Mimotope Immunotherapy Alleviates Allergic Responses in a Mouse Model.

Alternaria is a major outdoor allergen. Immunotherapy with Alternaria extracts has been documented to be effective in the sensitized patients. However, Alternaria extracts are notoriously difficult to standardize. Our aim is to screen the B cell mimotopes of Alternaria and to evaluate the therapeutic effects of B cell mimotope peptides on a BALB/c mouse model of Alternaria allergy. After a human sera pool from Alternaria monosensitized patients was established, B cell mimotopes were screened by a phage-displayed random heptamer peptide library that was identified via mixed Alternaria-specific IgE in the sera pool. B cell mimotopes with phage as a carrier were used to perform immunotherapy in an Alternaria allergy mouse model. Serological Ab levels, lung histology, and cytokine profiles were compared in the mimotope immunotherapy group, natural extract immunotherapy group, irrelevant phage control group, Alternaria-sensitized model group, and saline-blank group. Two mimotopes (MISTSRK and QKRNTIT) presented high binding ability with the sera of the Alternaria-allergic patients and mice and, therefore, were selected for immunotherapy in the mouse model. Compared with irrelevant phage control, model, and natural extract immunotherapy group, mimotope immunotherapy group significantly reduced serum IgE levels, inflammatory cells infiltration in the lung tissue, and IL-4 levels in bronchoalveolar lavage fluid, whereas serum IgG1 and IFN-γ levels in bronchoalveolar lavage fluid were increased. Our results indicate that B cell mimotopes of Alternaria alleviates allergic response in a mouse model and have potential as novel therapeutic agents for IgE-mediated Alternaria-allergic diseases.

Controlling Evolution: The Nobel Prize in Chemistry 2018.

2018 was a revolutionary year in the field of chemistry as the Royal Swedish Academy of Sciences awarded the Nobel Prize in Chemistry 2018 to three brilliant chemists (Figure 1): Dr. Frances H. Arnold-"for the directed evolution of enzymes "; Dr. George P. Smith and Sir Gregory P. Winter (jointly)-"for the phage display of peptides and antibodies" [1]. Their phenomenal contribution in controlling the evolution of enzymes and binding proteins and using them for the benefit of humankind has revolutionized the industry and academia alike for the past few decades.

Abstract 1147: A phage display identified peptide selectively binds to kidney injury molecule-1(KIM-1) and detects KIM-1-overexpressing tumors in vivo

Abstract Kidney injury molecule-1 (KIM-1), also known as hepatitis A virus cellular receptor-1 and T cell Ig mucin-1, is a transmembrane protein, which is not overexpressed in normal kidneys, but is significantly upregulated in damaged kidney tubular epithelial cells in toxic and ischemic acute kidney injury. Of interest, KIM-1 is overexpressed in most cases of renal cell carcinoma (RCC) and in other types of tumors including ovarian clear cell carcinoma and lung cancer. The extracellular domain of KIM-1 can be identified in the urine of patients with RCC and can thus be used as a biomarker for the detection of RCC. This study was carried out to identify a peptide that selectively binds to KIM-1 by screening a phage-displayed peptide library and to use the peptide for the detection of KIM-1-overexpressing tumors in vivo. Biopanning of a phage-displayed peptide library was performed on KIM-1-coated plates. Compared to other phage clones selected after biopanning, a phage clone displaying the CNWMINKEC peptide showed higher binding affinity to KIM-1 as examined by fluorometry and KIM-1-overexpressing 769-P renal tumor cells 1 as examined by ELISA. The CNWMINKEC peptide and a NeutrAvidin/biotin-CNWMINKEC multimer selectively bound to KIM-1 over albumin as examined by fluorometry and to KIM-1-overexpressing 769-P cells and A549 lung tumor cells compared to KIM-1-low expressing HEK293 normal cells when examined by flow cytometry. 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. 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. The CNWMINKEC peptide was not cytotoxic to cells and was stable for up to 24 h in the presence of serum. These results suggest that the CNWMINKEC peptide is a promising probe for in vivo imaging and detection of KIM-1-overexpressing tumors. Keywords In vivo imaging, kidney injury molecule-1, peptide, phage display, renal cell carcinoma Note: This abstract was not presented at the meeting. Citation Format: Jae-Won Yoon, Soo-woong Lee, Md. Enamul Haque, Rang- Woon Park, Moon-Chang Baek, Dong-Kyu Kim, Sang Kyoon Kim, Byungheon Lee. A phage display identified peptide selectively binds to kidney injury molecule-1(KIM-1) and detects KIM-1-overexpressing tumors in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1147.

An affinity peptide-incorporated electrochemical biosensor for the detection of neutrophil gelatinase-associated lipocalin

In this study, we demonstrate a novel affinity peptide-incorporated electrochemical biosensor for the detection of acute kidney injury and the diabetic biomarker neutrophil gelatinase-associated lipocalin (NGAL). Biopanning of the M13 phage display library over immobilized NGAL led to the rapid identification of unique affinity peptide with an amino acid sequence of DRWVARDPASIF, and the peptide-displayed phage particles were found to be specific affinities for NGAL. To address the development of peptide-based electrochemical sensor, a series of synthetic peptides away from phage particles was rationally designed, chemically synthesized, and immobilized to a gold sensor layer. Among five synthetic peptide derivatives tested, NGAL BP1 was selected as most promising recognition receptor, and its binding affinity was monitored by SWV and EIS. Using EIS, the limit of detection (LOD) was 1.74 ng/mL, while SWV had a LOD of 3.93 ng/mL. The detection performance of the peptide-incorporated sensor was comparable to commercially available ELISA NGAL detection kits. In addition, the validation of the peptide sensor was also confirmed with plasma from patients, and it was observed that the sensitivity of the peptide sensor showed a statistically significant difference. Our results show that the phage and peptide sensor system could detect NGAL with high sensitivity and selectivity, and this suggests its potential use as a biosensing platform for monitoring NGAL in a miniaturized electrochemical biosensor.

Panning using a phage-displayed random peptide library to identify peptides that antagonize the Helicobacter pylori ArsS acid-sensing domain

Helicobacter pylori is an important etiological factor involved in chronic gastritis, peptic ulcer, and gastric cancer. There are currently no optimal preventive or therapeutic interventions for H. pylori infection. H. pylori survives in the stomach by sensing and adapting to the highly acidic environment by using the two-component signal transduction system that contains the most widely known gastric acid receptor, ArsRS (which is composed of ArsS and ArsR). This study aimed to identify peptides that antagonize the acid-sensing domain of H. pylori ArsS. These peptides could be used to block the acid-sensing signal and thereby hinder H. pylori adaption to acidic environments to prevent its survival. Using proSite, the functional domains (including the N-terminal acid-sensing domain) of H. pylori J99 ArsS were predicted. The purified recombinant ArsS N-terminal acid-sensing protein (P-ArsS-A) was used as the target in a panning protocol in which peptides from the Ph.D.-7 Phage Display Peptide Library that could bind to P-ArsS-A were identified. As a result, eight phage clones that could specifically bind to P-ArsS-A were obtained and five amino acid sequences were identified, including P03 (MMSYPKH) and P06 (LTPMPNW). An in vitro minimum inhibitory concentration (MIC) evaluation showed that P03 and P06 significantly inhibited the growth of H. pylori J99. The MIC of P03 was 8 μM, and the MIC of P06 was >16 μM, indicating that P03 is a stronger inhibitor compared to P06. This was confirmed by colony counting on blood agar plates after P03 and P06 administration. Using homology modeling and molecular docking analysis, it was shown that P03 and P06 could bind to the ArsS N-terminal domain, and there were four shared binding sites: TYR25, ASN39, ARG73, and GLU74. Additionally, one hydrogen bond was found between P03 and ArsS, which is more cohesive than other forms of bonding (van der Waals force, other non-covalent bonds).

High-affinity phage-displayed peptide as a recognition probe for the detection of Cry2Ad2-3

Cry2A is widely used in transgenic crops in combination with Cry1A toxins. The sensitive and robust detection of Cry2A toxin in food and the environment is necessary to monitor the safety of biopesticides. Here, we describe an approach that involves the use of phage-displayed peptide for the detection of Cry2Ad2-3–the main area of Cry2Ad2 insecticidal activity. After four rounds of panning, six positive monoclonal phage particles were obtained. Pep5 with a sequence of ACSYNHNSKCGGG displayed low cross-reactivity with other Cry toxins. The working range of detection for Cry2Ad2-3 toxin standards in the brush border membrane vesicle (BBMV)–peptide sandwich ELISA was 10–50.625 ng mL−1 and the detection limit (LOD) was 8 ng mL−1. Molecular insight into the interaction of pep5 with Cry2Ad2-3 was gleaned using homology modeling and docking. Molecular docking results showed that high-affinity peptide tended to dock in the groove between the two domains of Cry2Ad2-3. The interactions within the toxin-pep5 complex were due to hydrogen bond and hydrophobic interaction. Pep5 also lead us to trap the binding region. Therefore, peptides may be a cost-efficient alternative for detecting Cry toxins and studying their mechanisms.

Cyclic peptide: a safe and effective alternative to synthetic aflatoxin B1-competitive antigens.

Aflatoxin B1 (AFB1) is one of the major mycotoxins, which naturally occurs in food and agricultural products. In this study, a cyclic peptide (CVPSKPGLC) mimicking AFB1 was used to develop a biotinylated peptide-based immunoassay (bp-ELISA) for AFB1 determination. This cyclic peptide was isolated from a commercially available phage-displayed random 7-mer cyclic peptide library, and then synthesized chemically. Instead of phage particles, the peptide was biotinylated and used to detect AFB1 by bp-ELISA, with an IC50 of 0.92ng/mL, which was approximately 60-fold better than that of phage ELISA. Good recoveries (83-102%) were obtained in spiked rice and corn samples, which were further validated by high-performance liquid chromatography-fluorescence detector. As better sensitivities (0.92-1.21ng/mL) were obtained by bp-ELISA even using selected anti-AFB1 antibodies prepared previously in laboratory, this cyclic peptide is suitable as a substitute for synthetic competitive AFB1 antigens in food contamination monitoring. Graphical abstract.

Genetically Encoded Libraries of Constrained Peptides.

Many therapeutic peptides can still be improved with respect to target specificity, target affinity, resistance to peptidases/proteases, physical stability, and capacity to pass through membranes required for oral delivery. Several modifications can improve the peptides' properties, in particular those that impose (a) conformational constraint(s). Screening of constrained peptides and the identification of hits is greatly facilitated by the generation of genetically encoded libraries. Recent breakthrough bacterial, phage, and yeast display screening systems of ribosomally synthesized post-translationally constrained peptides, particularly those of lanthipeptides, are earning special attention. Here we provide an overview of display systems for constrained, genetically encoded peptides and indicate prospects of constrained peptide-displaying phage and bacterial systems as such in vivo.

Phage-Displayed Peptide of Keratinocyte Growth Factor and Its Biological Effects on Epidermal Cells

Using monoclonal anti-human keratinocyte growth factor (KGF) antibody as the target in this study, we screened the phage display 7-mer peptide library to isolate key sequences of KGF. Through alignment analysis, the key sequences of KGF were selected and used to synthesize bioactive peptides. After purification, structural modification and fluorescence label, the phage model peptides of KGF were added to epidermal cells. CCK-8 assay, quantitative real-time PCR analysis and Western blot analysis were employed to evaluate the effect of the phage model peptides on epidermal cells. Among the 26 sequences isolated from a phage display 7-mer peptide library, only one sequence was chosen to generate bioactive peptide of KGF. The synthesized peptide could bind to epidermal cells, facilitate cellular proliferation, and promote both the mRNA expression and protein expression of KGF receptor. The result of this study is expected to promote wound healing by using phage-displayed KGF peptide, which can play biological effects on epidermal cells.

Structural Dissection and Optimization of a Cation–π–π Stacking System in Human Pregnancy-Related Serine Protease–Peptide Complex

Human pregnancy-related serine protease (PRSP) belongs to the high temperature requirement A (HTRA) family of homo-oligomeric and ATP-independent serine proteases, which plays an important role in human placental development and has been recognized as a potential therapeutic target for diverse diseases. Previously, we have successfully obtained the halogenated versions of HTRA peptide ligands via a rational design approach. In this study, we systematically investigate the solution complex structure of PRSP PDZ domain with its phage-displayed peptide ligand and identify a cation–π–π stacking system across the domain–peptide complex interface. The structural configuration, energetic property and molecular basis of the stacking system are examined in detail by using theoretical calculations and affinity assays. It is revealed that the stacking system is a noncovalent unit independent of other regions of the domain–peptide complex, which plays crucial role in conferring affinity and specificity to the complex binding. In addition, a coupled effect between cation–π and π–π interactions in the stacking system is observed; breaking of one interaction can influence another interaction substantially. As compared to other aromatic amino acids such as Tyr, Phe and His as well as neutral amino acid Ala, the wild-type peptide residue Trp is found as the best choice that can effectively stabilize the domain–peptide complex architecture.

Screening, Identification, and Characterization of an Affinity Peptide Specific to MT1-MMP and Its Application in Tumor Imaging.

Membrane type-1 matrix metalloproteinase (MT1-MMP) plays a crucial role in many physiological and pathological processes, especially in tumor invasion and metastasis. Bioimaging of this key molecule may find wide usage in various applications. MT-loop is a unique sequence of MT1-MMP and locates in the surface of the protein. In our previous studies, AF7p, an affinity peptide that targeting the MT-loop domain of MT1-MMP, was identified by screening a phage display (Ph.D.) peptide library. However, the target of AF7p is a synthetic sequence which lacked native conformation of the MT-loop region; thus, the binding affinity and specificity in reality may not be optimal. In this study, we considered the 3-dimensional (3-D) conformation of the MT-loop area in the MT1-MMP molecule and designed a novel strategy to screen the Ph.D. peptide library. The peptide we obtained showed a better binding affinity to WT-MT1-MMP than AF7p as observed through enzyme-linked immunosorbent assay (ELISA) and biolayer interferometry (BLI). The new peptide labeled and attached MT1-MMP expression cell lines HT1080 and did not show any toxicity to cells. Furthermore, for in vivo imaging, HT1080 tumor-bearing mice with higher MT1-MMP expression accumulated more Cy5.5-HS7 than mice with MT1-MMP low-expression cell lines A549 at tumor sites, and the half-life of HS7 was longer than that of AF7p, as confirmed by ex vivo imaging of the main organs. These results suggest the feasibility of using the subtraction biopanning strategy to screen the affinity peptide targeting MT-loop regions and HS7 is a superior probe for noninvasively imaging MT1-MMP expression in MT1-MMP-positive tumor models. It provides impetus for further studies to use HS7 in early diagnosis of tumors and in peptide-mediated drugs.

The 2018 Nobel Prize in Chemistry: phage display of peptides and antibodies.

One-half of the 2018 Nobel Prize in Chemistry was awarded jointly to George P. Smith and Sir Gregory P. Winter "for the phage display of peptides and antibodies". This feature article summarizes significant achievements leading to the development of phage display of peptides and antibodies, where a bacteriophage is genetically modified to display peptides and proteins, with the primary aim of producing new biopharmaceuticals. These significant achievements are proven to be useful for the development of phage-based bioassays and biosensors.

Rational Identification of a Colorectal Cancer Targeting Peptide through Phage Display

Colorectal cancer is frequently diagnosed at an advanced stage due to the absence of early clinical indicators. Hence, the identification of new targeting molecules is crucial for an early detection and development of targeted therapies. This study aimed to identify and characterize novel peptides specific for the colorectal cancer cell line RKO using a phage-displayed peptide library. After four rounds of selection plus a negative step with normal colorectal cells, CCD-841-CoN, there was an obvious phage enrichment that specifically bound to RKO cells. Cell-based enzyme-linked immunosorbent assay (ELISA) was performed to assess the most specific peptides leading to the selection of the peptide sequence CPKSNNGVC. Through fluorescence microscopy and cytometry, the synthetic peptide RKOpep was shown to specifically bind to RKO cells, as well as to other human colorectal cancer cells including Caco-2, HCT 116 and HCT-15, but not to the normal non-cancer cells. Moreover, it was shown that RKOpep specifically targeted human colorectal cancer cell tissues. A bioinformatics analysis suggested that the RKOpep targets the monocarboxylate transporter 1, which has been implicated in colorectal cancer progression and prognosis, proven through gene knockdown approaches and shown by immunocytochemistry co-localization studies. The peptide herein identified can be a potential candidate for targeted therapies for colorectal cancer.

Cell and Material‐Specific Phage Display Peptides Increase iPS‐MSC Mediated Bone and Vasculature Formation In Vivo

Biomimetically designed materials matching the chemical and mechanical properties of tissue support higher mesenchymal stem cell (MSC) adhesion. However, directing cell-specific attachment and ensuring uniform cell distribution within the interior of 3D biomaterials remain key challenges in healing critical sized defects. Previously, a phage display derived MSC-specific peptide (DPIYALSWSGMA, DPI) was combined with a mineral binding sequence (VTKHLNQISQSY, VTK) to increase the magnitude and specificity of MSC attachment to calcium-phosphate biomaterials in 2D. This study investigates how DPI-VTK influences quantity and uniformity of iPS-MSC mediated bone and vasculature formation in vivo. There is greater bone formation in vivo when iPS-MSCs are transplanted on bone-like mineral (BLM) constructs coated with DPI-VTK compared to VTK (p < 0.002), uncoated BLM (p < 0.037), acellular BLM/DPI-VTK (p < 0.003), and acellular BLM controls (p < 0.01). This study demonstrates, for the first time, the ability of non-native phage-display designed peptides to spatially control uniform cell distribution on 3D scaffolds and increase the magnitude and uniformity of bone and vasculature formation in vivo. Taken together, the study validates phage display as a novel technology platform to engineer non-native peptides with the ability to drive cell specific attachment on biomaterials, direct bone regeneration, and engineer uniform vasculature in vivo.

Novel Bi-Functional 14-mer Peptides with Both Ovarian Carcinoma Cells Targeting and Magnetic Fe₃O₄Nanoparticles Affinity.

Fe3O4 magnetic nanoparticles (Fe3O4-MNPs) have attracted much interest for their potential medical applications due to their desirable magnetic properties. However, their potential cytotoxicity, high RES clearance in circulation, and nonspecific distribution in tissue might be the main obstacles in practice. In the present study, a novel bi-functional 14-mer peptide with both ovarian carcinoma cells targeting and magnetic Fe3O4 nanoparticles affinity was designed and synthesized, and then a facile and effective modification method was developed to bestow the Fe3O4-MNPs with tumor-targeting capability via modification, using the bi-functional peptides. First, on the basis of a tumor-targeting 7-mer peptide QQTNWSL (Q-L) and another Fe3O4-MNPs-targeting 7-mer peptide TVNFKLY (T-Y)—screened by phage-displayed peptide libraries—two bi-functional 14-mer peptides sequenced as LSWNTQQ-YLKFNVT (abbreviated as LQ-YT) and QQTNWSL-YLKFNVT (QL-YT) were synthesized through combining the Q-L peptide and T-Y peptide in predetermined configurations. Their specificity for bonding with A2780 tumor cells and affinity for Fe3O4-MNPs were verified. Then the bi-functional 14-mer peptides were applied to modify the Fe3O4-MNPs. Results showed that both bi-functional 14-mer peptides could be conjugated to the Fe3O4-MNPs surface with high affinity. Immunofluorescence and Prussian blue staining assays indicated that the LQ-YT-modified Fe3O4-MNPs could specifically bond to A2780 tumor cells. In addition to our findings suggesting that more β-turns and random coils are conducive to increasing polypeptide surface area for binding and exposing the target group and bonding sites on LQ-YT to external targets, we demonstrated that the bi-functional 14-mer peptide has affinity for Fe3O4-MNPs, and that Fe3O4-MNPs, which was modified with a 14-mer peptide, could be bestowed with a targeting affinity for ovarian carcinoma cells.

Antibody response after hepatitis B vaccine boost mapped with peptide-phage display

Recombinant hepatitis B virus vaccines confer protection by eliciting specific antibodies against the hepatitis B surface antigen (HBsAg), known as anti-HBs. However, the performance of rapid anti-HBs diagnostic tests generates concerns regarding consistency. Novel indicators of protection might be developed by monitoring changes in targeted HBsAg-epitope profile after vaccination. In this work, we test the feasibility of our peptide-phage display platform in identifying B-cell epitopes targeted at different time-points after hepatitis B vaccination. We combined this platform with a unique approach for in silico analysis of enriched sequences. Serum samples collected from one single patient who had two boosting immunizations against hepatitis B virus were used in two-rounds of selection experiments. Five epitope candidates from HBsAg were identified in silico; most of them were previously reported in the literature. Our results suggest that the number of recognized HBsAg epitopes is related to the decrease of anti-HBs over time.

Epitope Mapping of Major Ragweed Allergen Amb a 1

Ragweed is a prominent cause of seasonal allergies. Thus far, information on IgE-binding sites of major allergen in ragweed pollen, Amb a 1, is very limited. A powerful experimental method to gain insights on the allergen epitopes is the selection of peptides from biological libraries that bind to anti-allergen antibodies. In this work, we aimed to map IgE epitopes of Amb a 1 using epitope-mimicking short peptides - mimotopes that were affinity-selected from phage-displayed random peptide libraries. The peptides weakly aligned with the Amb a 1 primary sequence, thus suggesting that the epitopes are conformational. When the peptides were mapped onto the surface of Amb a 1 homology model, the EpiSearch analysis predicted the location of four potential epitopic sites on surface patches centred at residues K104, S110, H214, and W312. The peptides matching to the predicted epitopes bound selectively to the IgE from pool of ragweed-allergic patients' sera and therefore represent mimetics of Amb a 1 IgE epitopes. The knowledge of IgE epitopes is a prerequisite for the rational design of molecular-based approaches to diagnosis and immunotherapy of allergic diseases.

Phage displayed mimotope peptide-based immunosensor for green and ultrasensitive detection of mycotoxin deoxynivalenol

Immunoassays play an important role in the monitoring and detection of mycotoxins in food. However, mycotoxin conjugates in the immunoassay system synthesized with toxic mycotoxin as raw material has seriously harmed the environment and human health during and after the performance. In this work, using mycotoxin deoxynivalenol (DON) as a model happen, a selected DON mimotope (phage-displayed peptide) that specifically binds to anti-DON antibody was served as a mimetic competing antigen in electrochemical immunosensor for ultrasensitive and green detection of DON. Under the optimized conditions, the mimotope peptide-based electrochemical immunosensor showed a dynamic range of 0.1–10,000 pg/ml and the limit detection (LOD) of 0.07 pg/ml. In addition, the accuracy and reproducibility of the established electrochemical immunosensor were evaluated by testing cross-reactivity and spiking DON negative samples. The result showed that mimotope peptide-based immunosensor has negligible cross-reactivity with other mycotoxins, and the average recovery of corn and wheat sample was 90.4%–118% and 101.3%–111%, respectively. Furthermore, the novel concept might provide potential applications to a general method for the immunosensor of various toxic small molecules.