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Sample records for technology phage display

  1. Potential of phage-displayed peptide library technology to identify functional targeting peptides

    Science.gov (United States)

    Krumpe, Lauren RH; Mori, Toshiyuki

    2010-01-01

    Combinatorial peptide library technology is a valuable resource for drug discovery and development. Several peptide drugs developed through phage-displayed peptide library technology are presently in clinical trials and the authors envision that phage-displayed peptide library technology will assist in the discovery and development of many more. This review attempts to compile and summarize recent literature on targeting peptides developed through peptide library technology, with special emphasis on novel peptides with targeting capacity evaluated in vivo. PMID:20150977

  2. Application of phage display technology in targeted therapy of breast cancer

    Institute of Scientific and Technical Information of China (English)

    Mian Kong; Junye Wang; Baojiang Li

    2013-01-01

    Phage display is a technology of gene expression and screening, it is widely used in the fields of defining antigenepitopes, signal transduction, genetic treatment, parasites research and tumor targeted therapy. Breast cancer is the mostcommon cancer in women, we can obtain peptides specially associated with breast cancer by using phage display technology,and this method has great potential in early diagnosis of breast cancer and development new targeted drugs.

  3. Phage Display Technology in Biomaterials Engineering: Progress and Opportunities for Applications in Regenerative Medicine.

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    Martins, Ivone M; Reis, Rui L; Azevedo, Helena S

    2016-11-18

    The field of regenerative medicine has been gaining momentum steadily over the past few years. The emphasis in regenerative medicine is to use various in vitro and in vivo approaches that leverage the intrinsic healing mechanisms of the body to treat patients with disabling injuries and chronic diseases such as diabetes, osteoarthritis, and degenerative disorders of the cardiovascular and central nervous system. Phage display has been successfully employed to identify peptide ligands for a wide variety of targets, ranging from relatively small molecules (enzymes, cell receptors) to inorganic, organic, and biological (tissues) materials. Over the past two decades, phage display technology has advanced tremendously and has become a powerful tool in the most varied fields of research, including biotechnology, materials science, cell biology, pharmacology, and diagnostics. The growing interest in and success of phage display libraries is largely due to its incredible versatility and practical use. This review discusses the potential of phage display technology in biomaterials engineering for applications in regenerative medicine.

  4. Display technology on filamentous phage in the search for anti-infective biological agents

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    Nelson Santiago Vispo

    2016-01-01

    Full Text Available Introduction: The causes of antibiotic resistance are complex. The phage display technology has been used mainly to produce monoclonal antibodies (MAbs and peptides directed against cancer or inflammatory disease targets. Today, this technology is recognized as a powerful tool for selecting novel peptides and antibodies that can bind to a wide range of antigens, ranging from whole cells to proteins and lipid targets. In this review, we highlight research that exploits the phage display technology to discover new drugs against infectious diseases, with a focus on antimicrobial peptides and antibodies. Methods: Basic and recent literature review was made, mainly focused on general aspects of phage display technology and the application in the search of new peptides or antibodies of pharmaceutical use to combat the infectious diseases transmitted by bacteria and virus. Results: Updated information on the selected topics is shown, with a guiding and practical approach aimed at researchers in the field of molecular biology to continue deepening the technology with special emphasis in the applications that have been developed in Cuba. Conclusions: Advances in methods of screening, manufacturing, and humanization technologies show that phage display technology can significantly contribute in the fight against clinically important pathogens.

  5. Preparation of single chain variable fragment of MG7 mAb by phage display technology

    Institute of Scientific and Technical Information of China (English)

    Zhao-Cai Yu; Jie Ding; Yong-Zhan Nie; Dai-Ming Fan; Xue-Yong Zhang

    2001-01-01

    AIM To develop the single chain variable fragment of MG7 murine anti-human gastric cancer monoclonal antibody using the phage display technology for obtaining a tumor-targeting mediator. METHODS mRNA was isolated from MG7-producing murine hybridoma cell line and converted into cDNA. The variable fragments of heavy and light chain were amplified separately and assembled into ScFv with a specially constructed DNA linker by PCR. The ScFvs DNA was ligated into the phagmid vector pCANTAB5E and the ligated sample was transformed into competent E. Coli TG1. The transformed cells were infected with M13K07 helper phage to form MG7 recombinant phage antibody library. The volume and recombinant rate of the library were evaluated by means of bacterial colony count and restriction analysis. After two rounds of panning with gastric cancer cell line KATOⅢ of highly expressing MG7binding antigen, the phage clones displaying ScFv of the antibody were selected by ELISA from the enriched phage clones. The antigen-binding affinity of the positive clone was detected by competition ELISA. HB2151 E. Coli was transfected with the positive phage clone demonstrated by competition ELISA for production of a soluble form of the MG7 ScFv. ELISA assay was used to detect the antigenbinding affinity of the soluble MG7 ScFv. Finally, the relative molecular mass of soluble MG7 ScFv was measured by SDS-PAGE. RESULTS The VH, VL and ScFv DNAs were about 340bp,320bp and 750bp, respectively. The volume of the library was up to 2 × 106 and 8 of 11 random clones were recombinants. Two phage clones could strongly compete with the original MG7 antibody for binding to the antigen expressed on KATO Ⅲ cells. Within 2 strong positive phage clones, the soluble MG7 ScFv from one clone was found to have the binding activity with KATO Ⅲ cells.SDS-PAGE showed that the relative molecular weight of soluble MG7 ScFv was 32. CONCLUSION The MG7 ScFv was successfully produced by phage antibody technology, which may

  6. [Peptide phage display in biotechnology and biomedicine].

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    Kuzmicheva, G A; Belyavskaya, V A

    2016-07-01

    To date peptide phage display is one of the most common combinatorial methods used for identifying specific peptide ligands. Phage display peptide libraries containing billions different clones successfully used for selection of ligands with high affinity and selectivity toward wide range of targets including individual proteins, bacteria, viruses, spores, different kind of cancer cells and variety of nonorganic targets (metals, alloys, semiconductors etc.) Success of using filamentous phage in phage display technologies relays on the robustness of phage particles and a possibility to genetically modify its DNA to construct new phage variants with novel properties. In this review we are discussing characteristics of the most known non-commercial peptide phage display libraries of different formats (landscape libraries in particular) and their successful applications in several fields of biotechnology and biomedicine: discovery of peptides with diagnostic values against different pathogens, discovery and using of peptides recognizing cancer cells, trends in using of phage display technologies in human interactome studies, application of phage display technologies in construction of novel nano materials.

  7. Engineering human interferon α1c/86D with phage display technology

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    马学军; 胡荣; 吕海; 魏开坤; 张丽兰; 薛水星; 侯云德

    1999-01-01

    Human interferon-α1c/86D (IFNα1c/86D) was functionally displayed on the surface of the filamentous bacteriophage using a phagemid vector system (pCANTAB5E). The key amino acid residues involved in the receptor binding were further defined with phage displayed 6-mer peptide library and two neutralizing antibodies against linear epitopes on the IFN-α1b, indicating that residues 30, 33, 34, (AB-loop) and residues 124, 126, 127 (D helix, DE-loop) were more critical than the adjacent residues for recognition of receptor. In addition, a cassette mutagenesis library was generated by fully randomizing the sequence of the four positions 29, 31, 32 and 35 in AB-loop, and used to select phage-IFN variants with WISH-hased panning method. Three phage-IFN variants were isolated to possess more antiviral activity in the range of 4—16-fold than parental phage-IFN after IPTG-induced soluble expression. The results suggest that phage displayed phage-IFN α1c/86D variants with increased specific activity might be obta

  8. Phage display: concept, innovations, applications and future.

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    Pande, Jyoti; Szewczyk, Magdalena M; Grover, Ashok K

    2010-01-01

    Phage display is the technology that allows expression of exogenous (poly)peptides on the surface of phage particles. The concept is simple in principle: a library of phage particles expressing a wide diversity of peptides is used to select those that bind the desired target. The filamentous phage M13 is the most commonly used vector to create random peptide display libraries. Several methods including recombinant techniques have been developed to increase the diversity of the library. On the other extreme, libraries with various biases can be created for specific purposes. For instance, when the sequence of the peptide that binds the target is known, its affinity and selectivity can be increased by screening libraries created with limited mutagenesis of the peptide. Phage libraries are screened for binding to synthetic or native targets. The initial screening of library by basic biopanning has been extended to column chromatography including negative screening and competition between selected phage clones to identify high affinity ligands with greater target specificity. The rapid isolation of specific ligands by phage display is advantageous in many applications including selection of inhibitors for the active and allosteric sites of the enzymes, receptor agonists and antagonists, and G-protein binding modulatory peptides. Phage display has been used in epitope mapping and analysis of protein-protein interactions. The specific ligands isolated from phage libraries can be used in therapeutic target validation, drug design and vaccine development. Phage display can also be used in conjunction with other methods. The past innovations and those to come promise a bright future for this field.

  9. Identification of small molecule binding sites within proteins using phage display technology.

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    Rodi, D. J.; Agoston, G. E.; Manon, R.; Lapcevich, R.; Green, S. J.; Makowski, L.; Biosciences Division; EntreMed Inc.; Florida State Univ.

    2001-11-01

    Affinity selection of peptides displayed on phage particles was used as the basis for mapping molecular contacts between small molecule ligands and their protein targets. Analysis of the crystal structures of complexes between proteins and small molecule ligands revealed that virtually all ligands of molecular weight 300 Da or greater have a continuous binding epitope of 5 residues or more. This observation led to the development of a technique for binding site identification which involves statistical analysis of an affinity-selected set of peptides obtained by screening of libraries of random, phage-displayed peptides against small molecules attached to solid surfaces. A random sample of the selected peptides is sequenced and used as input for a similarity scanning program which calculates cumulative similarity scores along the length of the putative receptor. Regions of the protein sequence exhibiting the highest similarity with the selected peptides proved to have a high probability of being involved in ligand binding. This technique has been employed successfully to map the contact residues in multiple known targets of the anticancer drugs paclitaxel (Taxol), docetaxel (Taxotere) and 2-methoxyestradiol and the glycosaminoglycan hyaluronan, and to identify a novel paclitaxel receptor [1]. These data corroborate the observation that the binding properties of peptides displayed on the surface of phage particles can mimic the binding properties of peptides in naturally occurring proteins. It follows directly that structural context is relatively unimportant for determining the binding properties of these disordered peptides. This technique represents a novel, rapid, high resolution method for identifying potential ligand binding sites in the absence of three-dimensional information and has the potential to greatly enhance the speed of development of novel small molecule pharmaceuticals.

  10. Selection of a peptide mimicking neutralization epitope of hepatitis E virus with phage peptide display technology

    Institute of Scientific and Technical Information of China (English)

    Ying Gu; Jun Zhang; Ying-Bing Wang; Shao-Wei Li; Hai-Jie Yang; Wen-Xin Luo; Ning-Shao Xia

    2004-01-01

    AIM: To select the peptide mimicking the neutralization epitope of hepatitis E virus which bound to non-type-specific and conformational monoclonal antibodies (mAbs) 8C11 and 8H3 fromed 7-peptide phage display library, and expressed the peptide recombinant with HBcAg in E.coli, and to observe whether the recombinant HBcAg could still form virus like particle (VLP) and to test the activation of the recombinant polyprotein and chemo-synthesized peptide that was selected by mAb 8H3.METHODS: 8C11 and 8H3 were used to screen for binding peptides through a 7-peptide phage display library. After 4rounds of panning, monoclonal phages were selected and sequenced. The obtained dominant peptide coding sequences was then synthesized and inserted into amino acid 78 to 83 of hepatitis B core antigen (HBcAg), and then expressed in E. coli. Activity of the recombinant proteins was detected by Western blotting, VLPs of the recombinant polyproteins were tested by transmission electron microscopy and binding activity of the chemo-synthesized peptide was confirmed by BIAcore biosensor.RESULTS: Twenty-one positive monoclonal phages (10for 8CL1, and 11 for 8H3) were selected and the inserted fragments were sequenced. The DNA sequence coding for the obtained dominant peptides 8C11 (N′-His-Pro-Thr-LeuLeu-Arg-Ile-C′, named 8C11A) and 8H3 (N′-Ser-Ile-LeuPro- Tyr-Pro-Tyr-C′, named 8H3A) were then synthesized and cloned to the HBcAg vector, then expressed in E. coli.The recombinant proteins aggregated into homodimer or polymer on SDS-PAGE, and could bind to mAb 8C11 and 8H3 in Western blotting. At the same time, the recombinant polyprotein could form virus like particles (VLPs), which could be visualized on electron micrograph. The dominant peptide 8H3A selected by mAb 8H3 was further chemosynthesized, and its binding to mAb 8H3 could be detected by BIAcore biosensor.CONCLUSION: These results implicate that conformational neutralizing epitope can be partially modeled by a short

  11. Pitfalls to avoid when using phage display for snake toxins.

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    Laustsen, Andreas Hougaard; Lauridsen, Line Præst; Lomonte, Bruno; Andersen, Mikael Rørdam; Lohse, Brian

    2017-02-01

    Antivenoms against bites and stings from snakes, spiders, and scorpions are associated with immunological side effects and high cost of production, since these therapies are still derived from the serum of hyper-immunized production animals. Biotechnological innovations within envenoming therapies are thus warranted, and phage display technology may be a promising avenue for bringing antivenoms into the modern era of biologics. Although phage display technology represents a robust and high-throughput approach for the discovery of antibody-based antitoxins, several pitfalls may present themselves when animal toxins are used as targets for phage display selection. Here, we report selected critical challenges from our own phage display experiments associated with biotinylation of antigens, clone picking, and the presence of amber codons within antibody fragment structures in some phage display libraries. These challenges may be detrimental to the outcome of phage display experiments, and we aim to help other researchers avoiding these pitfalls by presenting their solutions.

  12. Identification of peptide sequences that selectively bind to pentaerythritol trinitrate hemisuccinate-a surrogate of PETN, via phage display technology.

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    Kubas, George; Rees, William; Caguiat, Jonathan; Asch, David; Fagan, Diana; Cortes, Pedro

    2017-03-01

    The present research investigates the identification of amino acid sequences that selectively bind to a pentaerythritol tetranitrate (PETN) explosive surrogate. Through the use of a phage display technique and enzyme-linked immunosorbent assays (ELISA), a peptide library was tested against pentaerythritol trinitrate hemisuccinate (PETNH), a surrogate of PETN, to screen for those with amino acids having affinity toward the explosive. The results suggest that the library contains peptides selective to PETNH. Following three rounds of panning, clones were picked and tested for specificity toward PETNH. ELISA results from these samples show that each phage clone has some level of selectivity for binding to PETNH. The peptides from these clones have been sequenced and shown to contain certain common amino acid segments among them. This work represents a technological platform for identifying amino-acid sequences selective toward any bio-chem analyte of interest.

  13. A new peptide ligand for targeting human carbonic anhydrase IX, identified through the phage display technology.

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    Vasileios Askoxylakis

    Full Text Available UNLABELLED: Carbonic anhydrase IX (CAIX is a transmembrane enzyme found to be overexpressed in various tumors and associated with tumor hypoxia. Ligands binding this target may be used to visualize hypoxia, tumor manifestation or treat tumors by endoradiotherapy. METHODS: Phage display was performed with a 12 amino acid phage display library by panning against a recombinant extracellular domain of human carbonic anhydrase IX. The identified peptide CaIX-P1 was chemically synthesized and tested in vitro on various cell lines and in vivo in Balb/c nu/nu mice carrying subcutaneously transplanted tumors. Binding, kinetic and competition studies were performed on the CAIX positive human renal cell carcinoma cell line SKRC 52, the CAIX negative human renal cell carcinoma cell line CaKi 2, the human colorectal carcinoma cell line HCT 116 and on human umbilical vein endothelial cells (HUVEC. Organ distribution studies were carried out in mice, carrying SKRC 52 tumors. RNA expression of CAIX in HCT 116 and HUVEC cells was investigated by quantitative real time PCR. RESULTS: In vitro binding experiments of (125I-labeled-CaIX-P1 revealed an increased uptake of the radioligand in the CAIX positive renal cell carcinoma cell line SKRC 52. Binding of the radioligand in the colorectal carcinoma cell line HCT 116 increased with increasing cell density and correlated with the mRNA expression of CAIX. Radioligand uptake was inhibited up to 90% by the unlabeled CaIX-P1 peptide, but not by the negative control peptide octreotide at the same concentration. No binding was demonstrated in CAIX negative CaKi 2 and HUVEC cells. Organ distribution studies revealed a higher accumulation in SKRC 52 tumors than in heart, spleen, liver, muscle, intestinum and brain, but a lower uptake compared to blood and kidney. CONCLUSIONS: These data indicate that CaIX-P1 is a promising candidate for the development of new ligands targeting human carbonic anhydrase IX.

  14. Improved Binding Activity of Antibodies against Major Histocompatibility Complex Class I Chain-Related Gene A by Phage Display Technology for Cancer-Targeted Therapy

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    Achara Phumyen

    2012-01-01

    Full Text Available Major histocompatibility complex class I chain-related gene A (MICA is an NKG2D ligand that is over-expressed under cellular stress including cancer transformation and viral infection. High expression of MICA in cancer tissues or patients' sera is useful for prognostic or follow-up markers in cancer patients. In this study, phage display technology was employed to improve antigen-binding activities of anti-MICA monoclonal antibodies (WW2G8, WW6B7, and WW9B8. The 12 amino acid residues in the complementarity determining regions (CDRs on the V domain of the heavy chain CDR3 (HCDR3 of these anti-MICA antibodies were modified by PCR-random mutagenesis, and phages displaying mutated anti-MICA Fab were constructed. After seven rounds of panning, five clones of phages displaying mutant anti-MICA Fab which exhibited 3–7-folds higher antigen-binding activities were isolated. Two clones of the mutants (phage-displayed mutant Fab WW9B8.1 and phage-displayed mutant Fab WW9B8.21 were confirmed to have antigen-binding specificity for cell surface MICA proteins by flow cytometry. These phage clones are able to recognize MICA in a native form according to positive results obtained by indirect ELISA and flow cytometry. Thus, these phage particles could be potentially used for further development of nanomedicine specifically targeting cancer cells expressing MICA proteins.

  15. MimoDB: a New Repository for Mimotope Data Derived from Phage Display Technology

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    Xianlong Wang

    2010-11-01

    Full Text Available Peptides selected from phage-displayed random peptide libraries are valuable in two aspects. On one hand, these peptides are candidates for new diagnostics, therapeutics and vaccines. On the other hand, they can be used to predict the networks or sites of protein-protein interactions. MimoDB, a new repository for these peptides, was developed, in which 10,716 peptides collected from 571 publications were grouped into 1,229 sets. Besides peptide sequences, other important information, such as the target, template, library and complex structure, was also included. MimoDB can be browsed and searched through a user-friendly web interface. For computational biologists, MimoDB can be used to derive customized data sets and benchmarks, which are useful for new algorithm development and tool evaluation. For experimental biologists, their results can be searched against the MimoDB database to exclude possible target-unrelated peptides. The MimoDB database is freely accessible at http://immunet.cn/mimodb/.

  16. Targeting cytokines: production and characterization of anti-TNF-α scFvs by phage display technology.

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    Abdolalizadeh, Jalal; Nouri, Mohammad; Zolbanin, Jafar Majidi; Barzegari, Abolfazl; Baradaran, Behzad; Barar, Jaleh; Coukos, George; Omidi, Yadollah

    2013-01-01

    The antibody display technology (ADT) such as phage display (PD) has substantially improved the production of monoclonal antibodies (mAbs) and Ab fragments through bypassing several limitations associated with the traditional approach of hybridoma technology. In the current study, we capitalized on the PD technology to produce high affinity single chain variable fragment (scFv) against tumor necrosis factor-alpha (TNF- α), which is a potent pro-inflammatory cytokine and plays important role in various inflammatory diseases and malignancies. To pursue production of scFv antibody fragments against human TNF- α, we performed five rounds of biopanning using stepwise decreased amount of TNF-α (1 to 0.1 μ g), a semi-synthetic phage antibody library (Tomlinson I + J) and TG1 cells. Antibody clones were isolated and selected through enzyme-linked immunosorbent assay (ELISA) screening. The selected scFv antibody fragments were further characterized by means of ELISA, PCR, restriction fragment length polymorphism (RFLP) and Western blot analyses as well as fluorescence microscopy and flow cytometry. Based upon binding affinity to TNF-α , 15 clones were selected out of 50 positive clones enriched from PD in vitro selection. The selected scFvs displayed high specificity and binding affinity with Kd values at nm range to human TNF-α . The immunofluorescence analysis revealed significant binding of the selected scFv antibody fragments to the Raji B lymphoblasts. The effectiveness of the selected scFv fragments was further validated by flow cytometry analysis in the lipopolysaccharide (LPS) treated mouse fibroblast L929 cells. Based upon these findings, we propose the selected fully human anti-TNF-α scFv antibody fragments as potential immunotherapy agents that may be translated into preclinical/clinical applications.

  17. Methods for Selecting Phage Display Antibody Libraries.

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    Jara-Acevedo, Ricardo; Diez, Paula; Gonzalez-Gonzalez, Maria; Degano, Rosa Maria; Ibarrola, Nieves; Gongora, Rafael; Orfao, Alberto; Fuentes, Manuel

    2016-01-01

    The selection process aims sequential enrichment of phage antibody display library in clones that recognize the target of interest or antigen as the library undergoes successive rounds of selection. In this review, selection methods most commonly used for phage display antibody libraries have been comprehensively described.

  18. Antibody VH and VL recombination using phage and ribosome display technologies reveals distinct structural routes to affinity improvements with VH-VL interface residues providing important structural diversity.

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    Groves, Maria A T; Amanuel, Lily; Campbell, Jamie I; Rees, D Gareth; Sridharan, Sudharsan; Finch, Donna K; Lowe, David C; Vaughan, Tristan J

    2014-01-01

    In vitro selection technologies are an important means of affinity maturing antibodies to generate the optimal therapeutic profile for a particular disease target. Here, we describe the isolation of a parent antibody, KENB061 using phage display and solution phase selections with soluble biotinylated human IL-1R1. KENB061 was affinity matured using phage display and targeted mutagenesis of VH and VL CDR3 using NNS randomization. Affinity matured VHCDR3 and VLCDR3 library blocks were recombined and selected using phage and ribosome display protocol. A direct comparison of the phage and ribosome display antibodies generated was made to determine their functional characteristics.In our analyses, we observed distinct differences in the pattern of beneficial mutations in antibodies derived from phage and ribosome display selections, and discovered the lead antibody Jedi067 had a ~3700-fold improvement in KD over the parent KENB061. We constructed a homology model of the Fv region of Jedi067 to map the specific positions where mutations occurred in the CDR3 loops. For VL CDR3, positions 94 to 97 carry greater diversity in the ribosome display variants compared with the phage display. The positions 95a, 95b and 96 of VLCDR3 form part of the interface with VH in this model. The model shows that positions 96, 98, 100e, 100f, 100 g, 100h, 100i and 101 of the VHCDR3 include residues at the VH and VL interface. Importantly, Leu96 and Tyr98 are conserved at the interface positions in both phage and ribosome display indicating their importance in maintaining the VH-VL interface. For antibodies derived from ribosome display, there is significant diversity at residues 100a to 100f of the VH CDR3 compared with phage display. A unique deletion of isoleucine at position 102 of the lead candidate, Jedi067, also occurs in the VHCDR3.As anticipated, recombining the mutations via ribosome display led to a greater structural diversity, particularly in the heavy chain CDR3, which in turn

  19. Effective Optimization of Antibody Affinity by Phage Display Integrated with High-Throughput DNA Synthesis and Sequencing Technologies.

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    Dongmei Hu

    Full Text Available Phage display technology has been widely used for antibody affinity maturation for decades. The limited library sequence diversity together with excessive redundancy and labour-consuming procedure for candidate identification are two major obstacles to widespread adoption of this technology. We hereby describe a novel library generation and screening approach to address the problems. The approach started with the targeted diversification of multiple complementarity determining regions (CDRs of a humanized anti-ErbB2 antibody, HuA21, with a small perturbation mutagenesis strategy. A combination of three degenerate codons, NWG, NWC, and NSG, were chosen for amino acid saturation mutagenesis without introducing cysteine and stop residues. In total, 7,749 degenerate oligonucleotides were synthesized on two microchips and released to construct five single-chain antibody fragment (scFv gene libraries with 4 x 10(6 DNA sequences. Deep sequencing of the unselected and selected phage libraries using the Illumina platform allowed for an in-depth evaluation of the enrichment landscapes in CDR sequences and amino acid substitutions. Potent candidates were identified according to their high frequencies using NGS analysis, by-passing the need for the primary screening of target-binding clones. Furthermore, a subsequent library by recombination of the 10 most abundant variants from four CDRs was constructed and screened, and a mutant with 158-fold increased affinity (Kd = 25.5 pM was obtained. These results suggest the potential application of the developed methodology for optimizing the binding properties of other antibodies and biomolecules.

  20. Development of two murine antibodies against Neospora caninum using phage display technology and application on the detection of N. caninum.

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    Jinhua Dong

    Full Text Available Neosporosis, caused by an intracellular parasite, Neospora caninum, is an infectious disease primarily of cattle and dogs. It occurs worldwide and causes huge damages to dairy farms. In this study, we immunized mice with recombinant surface-associated protein 1 of N. caninum (rNcSAG1 and developed two novel monoclonal antibodies, A10 and H3, against NcSAG1 using phage-display technology. Both clones bound to purified rNcSAG1 and the half maximal inhibitory concentrations of A10 and H3 are 50 and 72 nM of rNcSAG1, respectively. In immunofluorescence assays, both A10 and H3 Fabs bound to N. caninum parasites. Direct detection of N. caninum parasites was developed firstly using an enzyme-linked immunosorbent assay (ELISA with A10 and H3. Binding of A10 and H3 antibodies to rNcSAG1 was also inhibited by some certain anti-N. caninum antibodies in the neosporosis-positive cattle sera, suggesting they might bind to the same epitopes of NcSAG1 with those anti-N. caninum antibodies of bovine. These antibodies were demonstrated to have a potential for monitoring the N. caninum parasites in a dairy farm, which may lead to protect livestock from parasite-infection.

  1. Nanoscale bacteriophage biosensors beyond phage display

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    Lee JW

    2013-10-01

    Full Text Available Jong-Wook Lee,1 Jangwon Song,1,2 Mintai P Hwang,1 Kwan Hyi Lee1,2 1Center for Biomaterials, Biomedical Research Institute, Korea Institute of Science and Technology, Seoul, Korea; 2Department of Biomedical Engineering, University of Science and Technology, Seoul, Korea Abstract: Bacteriophages are traditionally used for the development of phage display technology. Recently, their nanosized dimensions and ease with which genetic modifications can be made to their structure and function have put them in the spotlight towards their use in a variety of biosensors. In particular, the expression of any protein or peptide on the extraluminal surface of bacteriophages is possible by genetically engineering the genome. In addition, the relatively short replication time of bacteriophages offers researchers the ability to generate mass quantities of any given bacteriophage-based biosensor. Coupled with the emergence of various biomarkers in the clinic as a means to determine pathophysiological states, the development of current and novel technologies for their detection and quantification is imperative. In this review, we categorize bacteriophages by their morphology into M13-based filamentous bacteriophages and T4- or T7-based icosahedral bacteriophages, and examine how such advantages are utilized across a variety of biosensors. In essence, we take a comprehensive approach towards recent trends in bacteriophage-based biosensor applications and discuss their outlook with regards to the field of biotechnology. Keywords: biosensing, M13 bacteriophage, T4 bacteriophage, bacterial detection, Escherichia coli, SPR sensor

  2. Recognition of epoxy with phage displayed peptides.

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    Swaminathan, Swathi; Cui, Yue

    2013-07-01

    The development of a general approach for non-destructive chemical and biological functionalization of epoxy could expand opportunities for both fundamental studies and creating various device platforms. Epoxy shows unique electrical, mechanical, chemical and biological compatibility and has been widely used for fabricating a variety of devices. Phage display has emerged as a powerful method for selecting peptides that possess enhanced selectivity and binding affinity toward a variety of targets. In this letter, we demonstrate for the first time a powerful yet benign approach for identifying binding motifs to epoxy via comprehensively screened phage displayed peptides. Our results show that the epoxy can be selectively recognized with peptide-displaying phages. Further, along with the development of epoxy-based microstructures; recognition of the epoxy with phage displayed peptides can be specifically localized in these microstructures. We anticipate that these results could open up exciting opportunities in the use of peptide-recognized epoxy in fundamental biochemical recognition studies, as well as in applications ranging from analytical devices, hybrid materials, surface and interface, to cell biology. Copyright © 2013 Elsevier B.V. All rights reserved.

  3. Phage display for the generation of antibodies for proteome research, diagnostics and therapy.

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    Schirrmann, Thomas; Meyer, Torsten; Schütte, Mark; Frenzel, André; Hust, Michael

    2011-01-10

    Twenty years after its development, antibody phage display using filamentous bacteriophage represents the most successful in vitro antibody selection technology. Initially, its development was encouraged by the unique possibility of directly generating recombinant human antibodies for therapy. Today, antibody phage display has been developed as a robust technology offering great potential for automation. Generation of monospecific binders provides a valuable tool for proteome research, leading to highly enhanced throughput and reduced costs. This review presents the phage display technology, application areas of antibodies in research, diagnostics and therapy and the use of antibody phage display for these applications.

  4. Phage Display for the Generation of Antibodies for Proteome Research, Diagnostics and Therapy

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    Michael Hust

    2011-01-01

    Full Text Available Twenty years after its development, antibody phage display using filamentous bacteriophage represents the most successful in vitro antibody selection technology. Initially, its development was encouraged by the unique possibility of directly generating recombinant human antibodies for therapy. Today, antibody phage display has been developed as a robust technology offering great potential for automation. Generation of monospecific binders provides a valuable tool for proteome research, leading to highly enhanced throughput and reduced costs. This review presents the phage display technology, application areas of antibodies in research, diagnostics and therapy and the use of antibody phage display for these applications.

  5. Interaction Analysis through Proteomic Phage Display

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    Gustav N. Sundell

    2014-01-01

    Full Text Available Phage display is a powerful technique for profiling specificities of peptide binding domains. The method is suited for the identification of high-affinity ligands with inhibitor potential when using highly diverse combinatorial peptide phage libraries. Such experiments further provide consensus motifs for genome-wide scanning of ligands of potential biological relevance. A complementary but considerably less explored approach is to display expression products of genomic DNA, cDNA, open reading frames (ORFs, or oligonucleotide libraries designed to encode defined regions of a target proteome on phage particles. One of the main applications of such proteomic libraries has been the elucidation of antibody epitopes. This review is focused on the use of proteomic phage display to uncover protein-protein interactions of potential relevance for cellular function. The method is particularly suited for the discovery of interactions between peptide binding domains and their targets. We discuss the largely unexplored potential of this method in the discovery of domain-motif interactions of potential biological relevance.

  6. Nanoscale bacteriophage biosensors beyond phage display.

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    Lee, Jong-Wook; Song, Jangwon; Hwang, Mintai P; Lee, Kwan Hyi

    2013-01-01

    Bacteriophages are traditionally used for the development of phage display technology. Recently, their nanosized dimensions and ease with which genetic modifications can be made to their structure and function have put them in the spotlight towards their use in a variety of biosensors. In particular, the expression of any protein or peptide on the extraluminal surface of bacteriophages is possible by genetically engineering the genome. In addition, the relatively short replication time of bacteriophages offers researchers the ability to generate mass quantities of any given bacteriophage-based biosensor. Coupled with the emergence of various biomarkers in the clinic as a means to determine pathophysiological states, the development of current and novel technologies for their detection and quantification is imperative. In this review, we categorize bacteriophages by their morphology into M13-based filamentous bacteriophages and T4- or T7-based icosahedral bacteriophages, and examine how such advantages are utilized across a variety of biosensors. In essence, we take a comprehensive approach towards recent trends in bacteriophage-based biosensor applications and discuss their outlook with regards to the field of biotechnology.

  7. Nanoscale bacteriophage biosensors beyond phage display

    Science.gov (United States)

    Lee, Jong-Wook; Song, Jangwon; Hwang, Mintai P; Lee, Kwan Hyi

    2013-01-01

    Bacteriophages are traditionally used for the development of phage display technology. Recently, their nanosized dimensions and ease with which genetic modifications can be made to their structure and function have put them in the spotlight towards their use in a variety of biosensors. In particular, the expression of any protein or peptide on the extraluminal surface of bacteriophages is possible by genetically engineering the genome. In addition, the relatively short replication time of bacteriophages offers researchers the ability to generate mass quantities of any given bacteriophage-based biosensor. Coupled with the emergence of various biomarkers in the clinic as a means to determine pathophysiological states, the development of current and novel technologies for their detection and quantification is imperative. In this review, we categorize bacteriophages by their morphology into M13-based filamentous bacteriophages and T4- or T7-based icosahedral bacteriophages, and examine how such advantages are utilized across a variety of biosensors. In essence, we take a comprehensive approach towards recent trends in bacteriophage-based biosensor applications and discuss their outlook with regards to the field of biotechnology. PMID:24143096

  8. K-Ras(G12D)-selective inhibitory peptides generated by random peptide T7 phage display technology.

    Science.gov (United States)

    Sakamoto, Kotaro; Kamada, Yusuke; Sameshima, Tomoya; Yaguchi, Masahiro; Niida, Ayumu; Sasaki, Shigekazu; Miwa, Masanori; Ohkubo, Shoichi; Sakamoto, Jun-Ichi; Kamaura, Masahiro; Cho, Nobuo; Tani, Akiyoshi

    2017-03-11

    Amino-acid mutations of Gly(12) (e.g. G12D, G12V, G12C) of V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (K-Ras), the most promising drug target in cancer therapy, are major growth drivers in various cancers. Although over 30 years have passed since the discovery of these mutations in most cancer patients, effective mutated K-Ras inhibitors have not been marketed. Here, we report novel and selective inhibitory peptides to K-Ras(G12D). We screened random peptide libraries displayed on T7 phage against purified recombinant K-Ras(G12D), with thorough subtraction of phages bound to wild-type K-Ras, and obtained KRpep-2 (Ac-RRCPLYISYDPVCRR-NH2) as a consensus sequence. KRpep-2 showed more than 10-fold binding- and inhibition-selectivity to K-Ras(G12D), both in SPR analysis and GDP/GTP exchange enzyme assay. KD and IC50 values were 51 and 8.9 nM, respectively. After subsequent sequence optimization, we successfully generated KRpep-2d (Ac-RRRRCPLYISYDPVCRRRR-NH2) that inhibited enzyme activity of K-Ras(G12D) with IC50 = 1.6 nM and significantly suppressed ERK-phosphorylation, downstream of K-Ras(G12D), along with A427 cancer cell proliferation at 30 μM peptide concentration. To our knowledge, this is the first report of a K-Ras(G12D)-selective inhibitor, contributing to the development and study of K-Ras(G12D)-targeting drugs. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Oligopeptide M13 Phage Display in Pathogen Research

    Directory of Open Access Journals (Sweden)

    Michael Hust

    2013-10-01

    Full Text Available Phage display has become an established, widely used method for selection of peptides, antibodies or alternative scaffolds. The use of phage display for the selection of antigens from genomic or cDNA libraries of pathogens which is an alternative to the classical way of identifying immunogenic proteins is not well-known. In recent years several new applications for oligopeptide phage display in disease related fields have been developed which has led to the identification of various new antigens. These novel identified immunogenic proteins provide new insights into host pathogen interactions and can be used for the development of new diagnostic tests and vaccines. In this review we focus on the M13 oligopeptide phage display system for pathogen research but will also give examples for lambda phage display and for applications in other disease related fields. In addition, a detailed technical work flow for the identification of immunogenic oligopeptides using the pHORF system is given. The described identification of immunogenic proteins of pathogens using oligopeptide phage display can be linked to antibody phage display resulting in a vaccine pipeline.

  10. Phage display library screening for identification of interacting protein partners.

    Science.gov (United States)

    Addepalli, Balasubrahmanyam; Rao, Suryadevara; Hunt, Arthur G

    2015-01-01

    Phage display is a versatile high-throughput screening method employed to understand and improve the chemical biology, be it production of human monoclonal antibodies or identification of interacting protein partners. A majority of cell proteins operate in a concerted fashion either by stable or transient interactions. Such interactions can be mediated by recognition of small amino acid sequence motifs on the protein surface. Phage display can play a crucial role in identification of such motifs. This report describes the use of phage display for the identification of high affinity sequence motifs that could be responsible for interactions with a target (bait) protein.

  11. Fluorescent T7 display phages obtained by translational frameshift

    NARCIS (Netherlands)

    Slootweg, E.J.; Keller, H.J.H.G.; Hink, M.A.; Borst, J.W.; Bakker, J.; Schots, A.

    2006-01-01

    Lytic phages form a powerful platform for the display of large cDNA libraries and offer the possibility to screen for interactions with almost any substrate. To visualize these interactions directly by fluorescence microscopy, we constructed fluorescent T7 phages by exploiting the flexibility of pha

  12. Exploring the Secretomes of Microbes and Microbial Communities Using Filamentous Phage Display

    Directory of Open Access Journals (Sweden)

    Dragana eGagic

    2016-04-01

    Full Text Available Microbial surface and secreted proteins (the secretome contain a large number of proteins that interact with other microbes, host and/or environment. These proteins are exported by the coordinated activities of the protein secretion machinery present in the cell. A group of phage, called filamentous phage, have the ability to hijack the cellular protein secretion machinery in order to amplify and assemble via a secretion-like process. This ability has been harnessed in the use of filamentous phage of Escherichia coli in biotechnology applications, including screening large libraries of variants for binding to bait of interest, from tissues in vivo to pure proteins or even inorganic substrates. In this review we discuss the roles of secretome proteins in pathogenic and non-pathogenic bacteria and corresponding secretion pathways. We describe the basics of phage display technology and its variants applied to discovery of bacterial proteins that have functions of interest for bacterial colonization and pathogenesis, through filamentous phage display library screening. Published literature also shows that phage display is suitable for secretome protein display as a tool for identification immunogenic peptides and can be used for discovery of vaccine candidates. Secretome selection aided by next-generation sequence analysis can also be used for selective display of the secretome at a microbial community scale, the latter revealing the richness of secretome functions of interest and surprising versatility in filamentous phage display of secretome proteins from large number of Gram-negative as well as Gram-positive bacteria and archaea.

  13. Chemical posttranslational modification of phage-displayed peptides.

    Science.gov (United States)

    Ng, Simon; Tjhung, Katrina F; Paschal, Beth M; Noren, Christopher J; Derda, Ratmir

    2015-01-01

    Phage-displayed peptide library has fueled the discovery of novel ligands for diverse targets. A new type of phage libraries that displays not only linear and disulfide-constrained cyclic peptides but moieties that cannot be encoded genetically or incorporated easily by bacterial genetic machinery has emerged recently. Chemical posttranslational modification of phage library is one of the simplest approaches to encode nonnatural moieties. It confers the library with new functionality and makes it possible to select and evolve molecules with properties not found in the peptides, for instance, glycopeptides recognized by carbohydrate-binding protein and peptides with photoswitching capability. To this end, we describe the newly emerging techniques to chemically modify the phage library and quantify the efficiency of the reaction with a biotin-capture assay. Finally, we provide the methods to construct N-terminal Ser peptide library that allows site-selective modification of phage.

  14. Probing Tumor Microenvironment With In Vivo Phage Display

    Science.gov (United States)

    2014-10-01

    technology. A, Peptides found in the “Phage pool alone” group are listed in a descending order of frequency. Note that CISQERGESC (CIS) and CIFSGEGESC ( CIF ...expressing CISQERGESC (CIS: 0.6% of the recovered phage clones) and 202 phages expressing a relevant peptide CIFSGEGESC ( CIF : 0.2%) (Table 1A). The...term ended showed that CIS and CIF , which have very similar amino acid sequences, bind to cultured hb6011 CAFs especially to filopodia and fibrous

  15. Automated Detection of Conformational Epitopes Using Phage Display Peptide Sequences

    Directory of Open Access Journals (Sweden)

    Surendra S Negi

    2009-01-01

    Full Text Available Background: Precise determination of conformational epitopes of neutralizing antibodies represents a key step in the rational design of novel vaccines. A powerful experimental method to gain insights on the physical chemical nature of conformational epitopes is the selection of linear peptides that bind with high affinities to a monoclonal antibody of interest by phage display technology. However, the structural characterization of conformational epitopes from these mimotopes is not straightforward, and in the past the interpretation of peptide sequences from phage display experiments focused on linear sequence analysis to find a consensus sequence or common sequence motifs.Results: We present a fully automated search method, EpiSearch that predicts the possible location of conformational epitopes on the surface of an antigen. The algorithm uses peptide sequences from phage display experiments as input, and ranks all surface exposed patches according to the frequency distribution of similar residues in the peptides and in the patch. We have tested the performance of the EpiSearch algorithm for six experimental data sets of phage display experiments, the human epidermal growth factor receptor-2 (HER-2/neu, the antibody mAb Bo2C11 targeting the C2 domain of FVIII, antibodies mAb 17b and mAb b12 of the HIV envelope protein gp120, mAb 13b5 targeting HIV-1 capsid protein and 80R of the SARS coronavirus spike protein. In all these examples the conformational epitopes as determined by the X-ray crystal structures of the antibody-antigen complexes, were found within the highest scoring patches of EpiSearch, covering in most cases more than 50% residues of experimental observed conformational epitopes. Input options of the program include mapping of a single peptide or a set of peptides on the antigen structure, and the results of the calculation can be visualized on our interactive web server.Availability: Users can access the EpiSearch from our web

  16. Phage display-based strategies for cloning and optimization of monoclonal antibodies directed against human pathogens.

    Science.gov (United States)

    Clementi, Nicola; Mancini, Nicasio; Solforosi, Laura; Castelli, Matteo; Clementi, Massimo; Burioni, Roberto

    2012-01-01

    In the last two decades, several phage display-selected monoclonal antibodies (mAbs) have been described in the literature and a few of them have managed to reach the clinics. Among these, the anti-respiratory syncytial virus (RSV) Palivizumab, a phage-display optimized mAb, is the only marketed mAb directed against microbial pathogens. Palivizumab is a clear example of the importance of choosing the most appropriate strategy when selecting or optimizing an anti-infectious mAb. From this perspective, the extreme versatility of phage-display technology makes it a useful tool when setting up different strategies for the selection of mAbs directed against human pathogens, especially when their possible clinical use is considered. In this paper, we review the principal phage display strategies used to select anti-infectious mAbs, with particular attention focused on those used against hypervariable pathogens, such as HCV and influenza viruses.

  17. Phage display-derived human antibodies in clinical development and therapy.

    Science.gov (United States)

    Frenzel, André; Schirrmann, Thomas; Hust, Michael

    2016-10-01

    Over the last 3 decades, monoclonal antibodies have become the most important class of therapeutic biologicals on the market. Development of therapeutic antibodies was accelerated by recombinant DNA technologies, which allowed the humanization of murine monoclonal antibodies to make them more similar to those of the human body and suitable for a broad range of chronic diseases like cancer and autoimmune diseases. In the early 1990s in vitro antibody selection technologies were developed that enabled the discovery of "fully" human antibodies with potentially superior clinical efficacy and lowest immunogenicity. Antibody phage display is the first and most widely used of the in vitro selection technologies. It has proven to be a robust, versatile platform technology for the discovery of human antibodies and a powerful engineering tool to improve antibody properties. As of the beginning of 2016, 6 human antibodies discovered or further developed by phage display were approved for therapy. In 2002, adalimumab (Humira®) became the first phage display-derived antibody granted a marketing approval. Humira® was also the first approved human antibody, and it is currently the best-selling antibody drug on the market. Numerous phage display-derived antibodies are currently under advanced clinical investigation, and, despite the availability of other technologies such as human antibody-producing transgenic mice, phage display has not lost its importance for the discovery and engineering of therapeutic antibodies. Here, we provide a comprehensive overview about phage display-derived antibodies that are approved for therapy or in clinical development. A selection of these antibodies is described in more detail to demonstrate different aspects of the phage display technology and its development over the last 25 years.

  18. Ricin Detection Using Phage Displayed Single Domain Antibodies

    Directory of Open Access Journals (Sweden)

    Ellen R. Goldman

    2009-01-01

    Full Text Available Phage-displayed single domain antibodies (sdAb were compared to monomeric solubly expressed sdAb and llama polyclonal antibodies for the detection of ricin. SdAb are comprised of the variable domain derived from camelid heavy chain only antibodies (HcAb. Although HcAb lack variable light chains, they as well as their derivative sdAb are able to bind antigens with high affinity. The small size of sdAb (~16 kDa, while advantageous in many respects, limits the number of labels that can be incorporated. The ability to incorporate multiple labels is a beneficial attribute for reporter elements. Opportunely, sdAb are often selected using phage display methodology. Using sdAb displayed on bacteriophage M13 as the reporter element gives the potential for incorporating a very high number of labels. We have demonstrated the use of both sdAb and phage- displayed sdAb for the detection of ricin using both enzyme linked immunosorbent assays (ELISAs and Luminex fluid array assays. The phage-displayed sdAb led to five to ten fold better detection of ricin in both the ELISA and Luminex assays, resulting in limits of detection of 1 ng/mL and 64 pg/mL respectively. The phage-displayed sdAb were also dramatically more effective for the visualization of binding to target in nitrocellulose dot blot assays, a method frequently used for epitope mapping.

  19. Selective posttranslational modification of phage-displayed polypeptides

    Energy Technology Data Exchange (ETDEWEB)

    Tsao, Meng-Lin; Tian, Feng; Schultz, Peter

    2013-02-05

    The invention relates to posttranslational modification of phage-displayed polypeptides. These displayed polypeptides comprise at least one unnatural amino acid, e.g., an aryl-azide amino acid such as p-azido-L-phenylalanine, or an alkynyl-amino acid such as para-propargyloxyphenylalanine, which are incorporated into the phage-displayed fusion polypeptide at a selected position by using an in vivo orthogonal translation system comprising a suitable orthogonal aminoacyl-tRNA synthetase and a suitable orthogonal tRNA species. These unnatural amino acids advantageously provide targets for posttranslational modifications such as azide-alkyne [3+2]cycloaddition reactions and Staudinger modifications.

  20. Selective posttranslational modification of phage-displayed polypeptides

    Energy Technology Data Exchange (ETDEWEB)

    Tsao, Meng-Lin; Tian, Feng; Schultz, Peter

    2013-11-19

    The invention relates to posttranslational modification of phage-displayed polypeptides. These displayed polypeptides comprise at least one unnatural amino acid, e.g., an aryl-azide amino acid such as p-azido-L-phenylalanine, or an alkynyl-amino acid such as para-propargyloxyphenylalanine, which are incorporated into the phage-displayed fusion polypeptide at a selected position by using an in vivo orthogonal translation system comprising a suitable orthogonal aminoacyl-tRNA synthetase and a suitable orthogonal tRNA species. These unnatural amino acids advantageously provide targets for posttranslational modifications such as azide-alkyne [3+2] cycloaddition reactions and Staudinger modifications.

  1. Biochemical functionalization of peptide nanotubes with phage displayed peptides

    Science.gov (United States)

    Swaminathan, Swathi; Cui, Yue

    2016-09-01

    The development of a general approach for the biochemical functionalization of peptide nanotubes (PNTs) could open up existing opportunities in both fundamental studies as well as a variety of applications. PNTs are spontaneously assembled organic nanostructures made from peptides. Phage display has emerged as a powerful approach for identifying selective peptide binding motifs. Here, we demonstrate for the first time the biochemical functionalization of PNTs via peptides identified from a phage display peptide library. The phage-displayed peptides are shown to recognize PNTs. These advances further allow for the development of bifunctional peptides for the capture of bacteria and the self-assembly of silver particles onto PNTs. We anticipate that these results could provide significant opportunities for using PNTs in both fundamental studies and practical applications, including sensors and biosensors nanoelectronics, energy storage devices, drug delivery, and tissue engineering.

  2. Plasmids and packaging cell lines for use in phage display

    Science.gov (United States)

    Bradbury, Andrew M.

    2012-07-24

    The invention relates to a novel phagemid display system for packaging phagemid DNA into phagemid particles which completely avoids the use of helper phage. The system of the invention incorporates the use of bacterial packaging cell lines which have been transformed with helper plasmids containing all required phage proteins but not the packaging signals. The absence of packaging signals in these helper plasmids prevents their DNA from being packaged in the bacterial cell, which provides a number of significant advantages over the use of both standard and modified helper phage. Packaged phagemids expressing a protein or peptide of interest, in fusion with a phage coat protein such as g3p, are generated simply by transfecting phagemid into the packaging cell line.

  3. Phage Display Derived IgNAR V Region Binding Domains for Therapeutic Development.

    Science.gov (United States)

    Ubah, Obinna C; Barelle, Caroline J; Buschhaus, Magdalena J; Porter, Andrew J

    2016-01-01

    Phage display technology has revolutionized the science of drug discovery by transforming the generation and manipulation of ligands, such as antibody fragments, enzymes, and peptides. The basis of this technology is the expression of recombinant proteins or peptides fused to a phage coat protein, and subsequent isolation of ligands based on a variety of catalytic, physicochemical/binding kinetic and/or biological characteristics. An incredible number of diagnostic and therapeutic domains have been successfully isolated using phage display technology. The variable domain of the New Antigen Receptors (VNAR) found in cartilaginous fish, is also amenable to phage display selection. Whilst not an antibody, VNARs are unquestionable the oldest (450 million years), and smallest antigen binding, single-domains so far identified in the vertebrate kingdom. Their role as an integral part of the adaptive immune system of sharks has been well established, enhancing our understanding of the evolutionary origins of humoral immunity and the unusual but divergent ancestry of the VNARs themselves. VNARs exhibit remarkable physicochemical properties, such as small size, stability in extreme conditions, solubility, molecular flexibility, high affinity and selectivity for target. The purpose of this review is to illustrate the important role phage display has played in the isolation and characterization of potent therapeutic and diagnostic VNAR domains.

  4. Identification of Soft Matter Binding Peptide Ligands Using Phage Display.

    Science.gov (United States)

    Günay, Kemal Arda; Klok, Harm-Anton

    2015-10-21

    Phage display is a powerful tool for the selection of highly affine, short peptide ligands. While originally primarily used for the identification of ligands to proteins, the scope of this technique has significantly expanded over the past two decades. Phage display nowadays is also increasingly applied to identify ligands that selectively bind with high affinity to a broad range of other substrates including natural and biological polymers as well as a variety of low-molecular-weight organic molecules. Such peptides are of interest for various reasons. The ability to selectively and with high affinity bind to the substrate of interest allows the conjugation or immobilization of, e.g., nanoparticles or biomolecules, or generally, facilitates interactions at materials interfaces. On the other hand, presentation of peptide ligands that selectively bind to low-molecular-weight organic materials is of interest for the development of sensor surfaces. The aim of this article is to highlight the opportunities provided by phage display for the identification of peptide ligands that bind to synthetic or natural polymer substrates or to small organic molecules. The article will first provide an overview of the different peptide ligands that have been identified by phage display that bind to these "soft matter" targets. The second part of the article will discuss the different characterization techniques that allow the determination of the affinity of the identified ligands to the respective substrates.

  5. Construction of human antibody gene libraries and selection of antibodies by phage display.

    Science.gov (United States)

    Frenzel, André; Kügler, Jonas; Wilke, Sonja; Schirrmann, Thomas; Hust, Michael

    2014-01-01

    Antibody phage display is the most commonly used in vitro selection technology and has yielded thousands of useful antibodies for research, diagnostics, and therapy.The prerequisite for successful generation and development of human recombinant antibodies using phage display is the construction of a high-quality antibody gene library. Here, we describe the methods for the construction of human immune and naive scFv gene libraries.The success also depends on the panning strategy for the selection of binders from these libraries. In this article, we describe a panning strategy that is high-throughput compatible and allows parallel selection in microtiter plates.

  6. High throughput functional epitope mapping: revisiting phage display platform to scan target antigen surface.

    Science.gov (United States)

    Rojas, Gertrudis; Tundidor, Yaima; Infante, Yanelys Cabrera

    2014-01-01

    Antibody engineering must be accompanied by mapping strategies focused on identifying the epitope recognized by each antibody to define its unique functional identity. High throughput fine specificity determination remains technically challenging. We review recent experiences aimed at revisiting the oldest and most extended display technology to develop a robust epitope mapping platform, based on the ability to manipulate target-derived molecules (ranging from the whole native antigen to antigen domains and smaller fragments) on filamentous phages. Single, multiple and combinatorial mutagenesis allowed comprehensive scanning of phage-displayed antigen surface that resulted in the identification of clusters of residues contributing to epitope formation. Functional pictures of the epitope(s) were thus delineated in the natural context. Successful mapping of antibodies against interleukin-2, epidermal growth factor and its receptor, and vascular endothelial growth factor showed the versatility of these procedures, which combine the accuracy of site-directed mutagenesis with the high throughput potential of phage display.

  7. Phage display of peptide / major histocompatibility class I complexes

    DEFF Research Database (Denmark)

    Vest Hansen, N; Ostergaard Pedersen, L; Stryhn, A;

    2001-01-01

    Major histocompatibility complex class I (MHC-I) molecules sample peptides from the intracellular environment and present them to cytotoxic T cells (CTL). To establish a selection system, and, thereby, enable a library approach to identify the specificities involved (that of the MHC-I for peptides...... and subsequently that ot the T cell receptor for peptide-MHC-I complex), we have fused a single chain peptide-MHC-I complex to the phage minor coat protein, gpIII, and displayed it on filamentous phage. Expression of peptide-MHC-I complexes was shown with relevant conformation-specific monoclonal antibodies and...

  8. 基于噬菌体展示技术的赭曲霉毒素A高密度模拟表位的表达研究%High Density Expression of Ochratoxin A Mimotope Based on Phage Display Technology

    Institute of Scientific and Technical Information of China (English)

    陈兴龙; 徐玲; 刘仁荣; 裘雪梅; 朱立鑫

    2012-01-01

    Ochratoxin A (OTA) mimotope was displayed on phage surface. Two complementary oligonucleotides which contained OTA mimotope DNA sequences were cloned into vector pC89S4. Helper phage MOI (multiplicity of infection) and IPTG addition were optimized to gain phages with high density expression of OTA mimotope through phage display technology. The expression efficiency was measured by ELISA analysis. The result showed that high density OTA mimotope phages were obtained successfully. Helper phage MOI and IPTG concentration showed little influence the expression of OTA mimotope. However, IPTG addition at different time points had great effects on the expression of OTA mimotope. The expression efficiency was greatly improved after optimization.%通过噬菌体展示技术展示赭曲霉毒素A(ochratoxinA,OTA)模拟表位。将带有OTA模拟表位序列的寡核苷酸双链克隆至载体pC89-COTA。利用噬菌体展示技术,通过优化辅助噬菌体感染复数、IPTG加入的时间与剂量等培养条件获得表达有高密度OTA模拟表位的噬菌体。再通过酶联免疫吸附检测(ELISA)方法比较不同的条件下的表达效果,探索最优表达条件。结果表明:成功获得高密度表达OTA模拟表位的噬菌体,辅助噬菌体感染复数与IPTG加入量对模拟表位表达量影响不大,IPTG加入时间对模拟表位表达量有较大影响,优化后模拟表位表达量大大高于优化前。

  9. Phage display peptide library technology's application in the diagnosis and therapy of tumor%噬菌体展示肽库技术在肿瘤诊治研究中的应用

    Institute of Scientific and Technical Information of China (English)

    黄斌; 俞杨; 王自正

    2011-01-01

    噬菌体展示肽库技术是将高度多样性的多肽与噬菌体衣壳蛋白融合表达,呈现于噬菌体表面的多肽具有相对独立的空间结构,能与配体结合,从而筛选特异性分子表位,其已成为肿瘤诊治研究的重要手段和有力工具.筛选与肿瘤细胞或血管表面细胞特异结合的多肽作为核素载体,制成探针,可以对肿瘤进行早期诊断和转移灶的定位,还可以进行核素治疗;以多肽为基础的靶向药物,可以弥补化学药物在杀伤肿瘤细胞的同时也损伤正常组织和器官的弊端,使得肿瘤治疗进入一个新时代.%Phage display peptide library technology facilitates displaying peptides of high diversity on the surface of phage coat proteins,which with their independent space structure bind with ligands to screen the specific molecule epitopes.With the development of this technology,it becomes an effective and powerful tool in tumor research.As nuclide carrier,peptides screened from phage display library binding specifically with tumor cells and tumor blood vessels,can be manufactured into a probe for prophase diagnosis of tumor,localization of metastasis and nuclide therapy.Targeting chemotherapy drugs on the basis of peptides greatly lower the risk of killing normal tissue and organs,which impulses entering a new therapy time.

  10. Heterogeneous catalysis on the phage surface: Display of active human enteropeptidase.

    Science.gov (United States)

    Gasparian, Marine E; Bobik, Tatyana V; Kim, Yana V; Ponomarenko, Natalia A; Dolgikh, Dmitry A; Gabibov, Alexander G; Kirpichnikov, Mikhail P

    2013-11-01

    Enteropeptidase (EC 3.4.21.9) plays a key role in mammalian digestion as the enzyme that physiologically activates trypsinogen by highly specific cleavage of the trypsinogen activation peptide following the recognition sequence D4K. The high specificity of enteropeptidase makes it a powerful tool in modern biotechnology. Here we describe the application of phage display technology to express active human enteropeptidase catalytic subunits (L-HEP) on M13 filamentous bacteriophage. The L-HEP/C122S gene was cloned in the g3p-based phagemid vector pHEN2m upstream of the sequence encoding the phage g3p protein and downstream of the signal peptide-encoding sequence. Heterogeneous catalysis of the synthetic peptide substrate (GDDDDK-β-naphthylamide) cleavage by phage-bound L-HEP was shown to have kinetic parameters similar to those of soluble enzyme, with the respective Km values of 19 μM and 20 μM and kcat of 115 and 92 s(-1). Fusion proteins containing a D4K cleavage site were cleaved with phage-bound L-HEP/C122S as well as by soluble L-HEP/C122S, and proteolysis was inhibited by soybean trypsin inhibitor. Rapid large-scale phage production, one-step purification of phage-bound L-HEP, and easy removal of enzyme activity from reaction samples by PEG precipitation make our approach suitable for the efficient removal of various tag sequences fused to the target proteins. The functional phage display technology developed in this study can be instrumental in constructing libraries of mutants to analyze the effect of structural changes on the activity and specificity of the enzyme or generate its desired variants for biotechnological applications.

  11. Identification of keratinocyte-specific markers using phage display and mass spectrometry

    DEFF Research Database (Denmark)

    Jensen, Kim Bak; Jensen, Ole Nørregaard; Ravn, Peter

    2003-01-01

    Specific molecular markers for various normal and pathogenic cell states and cell types provide knowledge of basic biological systems and have a direct application in targeted therapy. We describe a proteomic method based on the combination of new and improved phage display antibody technologies...... and mass spectrometry that allows identification of cell type-specific protein markers. The most important features of the method are (i) reduction of experimental noise originating from background binding of phage particles and (ii) isolation of affinity binders after a single round of selection, which...

  12. Phage Display-based Strategies for Cloning and Optimization of Monoclonal Antibodies Directed against Human Pathogens

    Directory of Open Access Journals (Sweden)

    Roberto Burioni

    2012-07-01

    Full Text Available In the last two decades, several phage display-selected monoclonal antibodies (mAbs have been described in the literature and a few of them have managed to reach the clinics. Among these, the anti-respiratory syncytial virus (RSV Palivizumab, a phage-display optimized mAb, is the only marketed mAb directed against microbial pathogens. Palivizumab is a clear example of the importance of choosing the most appropriate strategy when selecting or optimizing an anti-infectious mAb. From this perspective, the extreme versatility of phage-display technology makes it a useful tool when setting up different strategies for the selection of mAbs directed against human pathogens, especially when their possible clinical use is considered. In this paper, we review the principal phage display strategies used to select anti-infectious mAbs, with particular attention focused on those used against hypervariable pathogens, such as HCV and influenza viruses.

  13. Recombinant human antibody fragment against tetanus toxoid produced by phage display

    Science.gov (United States)

    Neelakantam, B.; Sridevi, N. V.; Shukra, A. M.; Sugumar, P.; Samuel, S.

    2014-01-01

    Phage display technology is a powerful in vitro method for the identification of specific monoclonal antibodies (antibody fragments) to an antigenic target and allows the rapid generation and selection of high affinity, fully human antibodies directed toward any disease target appropriate for antibody therapy. In the present study, we exploited the phage display technology for the selection of an antigen binding fragment (Fabs) toward tetanus toxoid using human naïve phage antibody library constructed from peripheral blood lymphocytes of naïve human donors. The phages displaying Fab were subjected to three rounds of bio-panning with tetanus toxoid as antigen on a solid phase. The high affinity antibody fragments were expressed in HB2151 strain of Escherichia coli and purified by immobilized metal affinity chromatography. The binding activity and specificity of the antibody fragment was established by its reactivity toward tetanus toxoid and non-reactivity toward other related toxins as determined by enzyme-linked immunosorbent assay and immunoblot analysis. The selected Fab fragment forming the antigen-binding complexes with the toxoid in flocculation assay indicates that the Fab may have a potential neutralizing ability toward antigen. PMID:24678405

  14. Selection of peptide ligands for the antimucin core antibody C595 using phage display technology: definition of candidate epitopes for a cancer vaccine.

    Science.gov (United States)

    Laing, P; Tighe, P; Kwiatkowski, E; Milligan, J; Price, M; Sewell, H

    1995-06-01

    Aims-To further define the specificity of the antimucin core antibody C595 by fitting it with a family of hexapeptide ligands by immunoselection of filamentous bacteriophage from a gene III display library of approximately 6.4 x 10(7) random hexapeptides.Methods-Three rounds of immuno-selection were used to enrich for C595 binding phage. DNA sequencing revealed the hexapeptides expressed. Bacteriophage and corresponding synthetic hexapeptides were used in ELISA assay to determine binding affinities.Results-Twenty nine clones from this selected population were analysed. Seven contained the natural epitope RPAP, encoded by two different DNA sequences; 17/29 contained the motif RLPP. In all, 28/29 clones contained the motif RXXP and one clone (RVRPAP) contained the motif RXXP in two peptidic registers; 24/28 clones (6/8 DNA sequences) contained a hydrophobic residue (V or I) at position 1 relative to the RXXP motif. In addition the proximity of RXXP to glycine (position 5) suggests that this contributes in the natural epitope to antibody/antigen binding, which was not detected by chemical synthetic methods. One clone, KSKAGV, bears no obvious relationship to the natural epitope and therefore qualifies as a weakly binding mimotope.Conclusions-This approach has rapidly defined the specificity of this antibody in unprecedented detail, and provides a more comprehensive molecular basis for exploring the immune recognition of the MUC1 mucin by the C595 antibody. Importantly, the novel but related epitopes seen provide peptide specificities and a strategy which may prove useful in generating cancer vaccine candidates.

  15. 噬菌体展示技术在血液肿瘤诊断治疗中的应用%The Application of Phage Display Technology in the Diagnosis and Therapy of Hematological Malignancies

    Institute of Scientific and Technical Information of China (English)

    王沂; 张春明; 郝建秀; 宋娜玲; 刘金剑; 贺欣; 刘鉴峰; 王德芝

    2012-01-01

    Hematopoietic malignancies are serious hazard to public health. Presently, the most ideal method in diagnosing and treating hematopoietic malignancies is specific molecular diagnosis and targeted therapy. However, the most stubborn difficulty of this method lies in the screening of molecular target. Phage display technology, a powerful approach developed in recent decade, has many advantages, including high throughput screening, mimicking natural epitopes, easy purification, integrating protein function with its coding gene, etc. Phage display technology is widely used in the area of molecular biology, such as screening of functional peptides and proteins, the study of protein interactions, identification of antigens, screening of genetically engineered antibodies, etc, which is very fit for the screening of ideal targets. At present, the application of this technology in hematopoietic malignancies diagnosis and therapy is -focus on phage antibody library and phage random peptide library. This article will introduce the research findings of phage display technology in hematopoietic malignancies diagnosis and therapy, and look forward to the prospect of this technology in this area.%血液肿瘤即造血系统的恶性肿瘤,是一种严重危害公共健康的疾病.目前,血液肿瘤诊断治疗的最理想方法就是分子特异性诊断和靶向治疗,但该方法面临的最大困难就是分子靶点的选择.噬菌体展示技术是近十年发展起来的一种新的生物学技术,具有高通量筛选、模拟天然表位、易于纯化、将蛋白功能与编码基因相统一等优点,广泛应用于功能性蛋白质和多肽的筛选、蛋白质间的识别与相互作用、抗原表位的鉴定、基因工程抗体的筛选等多个分子生物学领域,非常适于理想靶点的选择.目前,噬菌体文库技术在血液肿瘤诊治中的应用主要集中在噬菌体抗体文库和噬菌体随机放库上.本文就噬菌体展示技术在血液

  16. MIMOX: a web tool for phage display based epitope mapping

    Directory of Open Access Journals (Sweden)

    Honda Wataru

    2006-10-01

    Full Text Available Abstract Background Phage display is widely used in basic research such as the exploration of protein-protein interaction sites and networks, and applied research such as the development of new drugs, vaccines, and diagnostics. It has also become a promising method for epitope mapping. Research on new algorithms that assist and automate phage display based epitope mapping has attracted many groups. Most of the existing tools have not been implemented as an online service until now however, making it less convenient for the community to access, utilize, and evaluate them. Results We present MIMOX, a free web tool that helps to map the native epitope of an antibody based on one or more user supplied mimotopes and the antigen structure. MIMOX was coded in Perl using modules from the Bioperl project. It has two sections. In the first section, MIMOX provides a simple interface for ClustalW to align a set of mimotopes. It also provides a simple statistical method to derive the consensus sequence and embeds JalView as a Java applet to view and manage the alignment. In the second section, MIMOX can map a single mimotope or a consensus sequence of a set of mimotopes, on to the corresponding antigen structure and search for all of the clusters of residues that could represent the native epitope. NACCESS is used to evaluate the surface accessibility of the candidate clusters; and Jmol is embedded to view them interactively in their 3D context. Initial case studies show that MIMOX can reproduce mappings from existing tools such as FINDMAP and 3DEX, as well as providing novel, rational results. Conclusion A web-based tool called MIMOX has been developed for phage display based epitope mapping. As a publicly available online service in this area, it is convenient for the community to access, utilize, and evaluate, complementing other existing programs. MIMOX is freely available at http://web.kuicr.kyoto-u.ac.jp/~hjian/mimox.

  17. Construction and selection of the natural immune Fab antibody phage display library from patients with colorectal cancer

    Institute of Scientific and Technical Information of China (English)

    Bao-Ping Wu; Bing Xiao; Tian-Mo Wan; Ya-Li Zhang; Zhen-Shu Zhang; Dian-Yuan Zhou; Zhuo-Sheng Lai; Chun-Fang Gao

    2001-01-01

    AIM: To construct the natural immune Fab antibody phage display libraries of colorectal cancer and to select antibodies related with colorectal cancer. METHODS: Extract total RNA from tissue of local cancer metastasis lymph nodes of patients with colorectal cancer.RT-PCR was used to amplify the heavy chain Fd and light chain к and the amplification products were inserted successively into the vector pComb3 to construct the human libraries of Fab antibodies. They were then panned by phage display technology. By means of Dot immunoblotting and ELISA, the libraries were identified and the Fab phage antibodies binding with antigens of colorectal cancer were selected. RESULTS: The amplified fragments of Fd and к gained by RT-PCR were about 650bp. Fd and к PCR products were subsequently inserted into the vector pComb3, resulting in a recombination rate of 40% and the volume of Fab phage display library reached 1.48 x 106. The libraries were enriched about 120-fold by 3 cycles of adsorption-elution- multiplication (panning). Dot immunoblotting showed Fab expressions on the phage libraries and ELISA showed 5clones of Fab phage antibodies which had binding activities with antigens of colorectal cancer. CONCLUSION: The natural immune Fab antibody phage display libraries of colorectal cancer were constructed. They could be used to select the relative antibodies of colorectal cancer.

  18. Identification of gliadin-binding peptides by phage display

    Directory of Open Access Journals (Sweden)

    Östman Sofia

    2011-02-01

    Full Text Available Abstract Background Coeliac disease (CD is a common and complex disorder of the small intestine caused by intolerance to wheat gluten and related edible cereals like barley and rye. Peptides originating from incomplete gliadin digestion activate the lamina propria infiltrating T cells to release proinflammatory cytokines, which in turn cause profound tissue remodelling of the small intestinal wall. There is no cure for CD except refraining from consuming gluten-containing products. Results Phage from a random oligomer display library were enriched by repeated pannings against immobilised gliadin proteins. Phage from the final panning round were plated, individual plaques picked, incubated with host bacteria, amplified to a population size of 1011 to 1012 and purified. DNA was isolated from 1000 purified phage populations and the region covering the 36 bp oligonucleotide insert from which the displayed peptides were translated, was sequenced. Altogether more than 150 different peptide-encoding sequences were identified, many of which were repeatedly isolated under various experimental conditions. Amplified phage populations, each expressing a single peptide, were tested first in pools and then one by one for their ability to inhibit binding of human anti-gliadin antibodies in ELISA assays. These experiments showed that several of the different peptide-expressing phage tested inhibited the interaction between gliadin and anti-gliadin antibodies. Finally, four different peptide-encoding sequences were selected for further analysis, and the corresponding 12-mer peptides were synthesised in vitro. By ELISA assays it was demonstrated that several of the peptides inhibited the interaction between gliadin molecules and serum anti-gliadin antibodies. Moreover, ELISA competition experiments as well as dot-blot and western blot revealed that the different peptides interacted with different molecular sites of gliadin. Conclusions We believe that several of

  19. Phage display screen for peptides that bind Bcl-2 protein.

    Science.gov (United States)

    Park, Hye-Yeon; Kim, Joungmok; Cho, June-Haeng; Moon, Ji Young; Lee, Su-Jae; Yoon, Moon-Young

    2011-01-01

    Bcl-2 family proteins are key regulators of apoptosis associated with human disease, including cancer. Bcl-2 protein has been found to be overexpressed in many cancer cells. Therefore, Bcl-2 protein is a potential diagnostic target for cancer detection. In the present study, the authors have identified several Bcl-2 binding peptides with high affinity (picomolar range) from a 5-round M13 phage display library screening. These peptides can be used to develop novel diagnostic probes or potent inhibitors with diverse polyvalencies.

  20. Handbook of display technology

    CERN Document Server

    Castellano, Joseph A

    1992-01-01

    This book presents a comprehensive review of technical and commercial aspects of display technology. It provides design engineers with the information needed to select proper technology for new products. The book focuses on flat, thin displays such as light-emitting diodes, plasma display panels, and liquid crystal displays, but it also includes material on cathode ray tubes. Displays include a large number of products from televisions, auto dashboards, radios, and household appliances, to gasoline pumps, heart monitors, microwave ovens, and more.For more information on display tech

  1. SELECTION OF NEW EPITOPES FROM MONOVALENT DISPLAYED PHAGE OCTAPEPTIDE LIBRARY

    Institute of Scientific and Technical Information of China (English)

    李全喜; 王琰; 李竞; 王雅明; 徐建军; 王力民; 董志伟

    1998-01-01

    A library of 2×l07 random oetspaptides was constructed by use of phegemid-based monovaient phage display system. The randomly synthesized degenerated oilgodeoxyribonucleotides (oligos) were fused to the truncated gⅢ (p210-p408). Sequeraze analysis of 11 randomly chosen clones suggested that the degenerated inserts and its deduced amino acid (an) sequences are randomly distributed. The library was used to select binding paptides to the morroeloncl antlhody (mAb) 9E10, which recognizes a continuous decapaptide epitope of denatured human c-myc protein. After four to five rounds of panning, most of the eluted clones could bind to 9E10. Sequerlce analysis of the selected positive clones indlcated that the binding sequences could fall into two chsses, one class (clone 1) shares a consensus motif, ISE x x L, with c-mire decapeprider and the sequences of the other class are entirely different. The binding of both classes to 9E10 could be specifically lnhlhited by froe c-myc deeapeptide. The immunogenlcitF cff the phage peptide was further investigsted h5, construction of multivalent displayed phage peptides and immunization of animals with or without adjuvant. ELISA and competitive ELISA showed that anti-serum from both mice and rabbit immunized with either done could bind to the original antigen, c-myc decapeptide. These results denote that in spite of the dissimilarity of the selected psptides with c-myc decapeptide, they are capable of inducing similar immune respones in vivo, thus actually mimicking the antigen epitope.

  2. Screening for PreS specific binding ligands with a phage displayed peptides library

    Institute of Scientific and Technical Information of China (English)

    Qiang Deng; Ming Zhuang; Yu-Ying Kong; You-Hua Xie; Yuan Wang

    2005-01-01

    AIM: To construct a random peptide phage display library and search for peptides that specifically bind to the PreS region of hepatitis B virus (HBV).METHODS: A phage display vector, pFuse8, based on the gene 8 product (pⅧ) of M13 phage was made and used to construct a random peptide library. E. coli derived thioredoxin-PreS was purified with Thio-bond beads, and exploited as the bait protein for library screening. Five rounds of bio-panning were performed. The PreS-binding specificities of enriched phages were characterized with phage ELISA assay.RESULTS: A phage display vector was successfully constructed as demonstrated to present a pⅧ fused HBV PreS1 epitope on the phage surface with a high efficiency.A cysteine confined random peptide library was constructed containing independent clones exceeding 5±108 clone forming unit (CFU). A pool of phages showing a PreS-binding specificity was obtained after the screening against thioPres with an enrichment of approximately 400 times. Five phages with high PreS-binding specificities were selected and characterized. Sequences of the peptides displayed on these phages were determined.CONCLUSION: A phage library has been constructed,with random peptides displaying as pⅧ-fusion proteins.Specific PreS-binding peptides have been obtained, which may be useful for developing antivirals against HBV infection.

  3. Raising an Antibody Specific to Breast Cancer Subpopulations Using Phage Display on Tissue Sections

    DEFF Research Database (Denmark)

    Larsen, Simon Asbjørn; Meldgaard, Theresa; Fridriksdottir, Agla Jael Rubner

    2016-01-01

    BACKGROUND/AIM: Primary tumors display a great level of intra-tumor heterogeneity in breast cancer. The current lack of prognostic and predictive biomarkers limits accurate stratification and the ability to predict response to therapy. The aim of the present study was to select recombinant antibody...... fragments specific against breast cancer subpopulations, aiding the discovery of novel biomarkers. MATERIALS AND METHODS: Recombinant antibody fragments were selected by phage display. A novel shadowstick technology enabled the direct selection using tissue sections of antibody fragments specific against...... small subpopulations of breast cancer cells. Selections were performed against a subpopulation of breast cancer cells expressing CD271(+), as these previously have been indicated to be potential breast cancer stem cells. The selected antibody fragments were screened by phage ELISA on both breast cancer...

  4. Screening of TACE Peptide Inhibitors from Phage Display Peptide Library

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    To obtain the recombinant tumor necrosis factor-α converting enzyme (TACE) ectodomain and use it as a selective molecule for the screening of TACE peptide inhibitors, the cDNA coding catalytic domain (T800) and full-length ectodomain (T1300) of TACE were amplified by RTPCR, and the expression plasmids were constructed by inserting T800 and T1300 into plasmid pET28a and pET-28c respectively. The recombinant T800 and T1300 were induced by IPTG, and SDSPAGE and Western blotting analysis results revealed that T800 and T1300 were highly expressed in the form of inclusion body. After Ni2+-NTA resin affinity chromatography, the recombinant proteins were used in the screening of TACE-binding peptides from phage display peptide library respectively. After 4 rounds of biopanning, the positive phage clones were analyzed by ELISA, competitive inhibition assay and DNA sequencing. A common amino acid sequence (TRWLVYFSRPYLVAT) was found and synthesized. The synthetic peptide could inhibit the TNF-α release from LPS-stimulated human peripheral blood mononuclear cells (PBMC) up to 60.3 %. FACS analysis revealed that the peptide mediated the accumulation of TNF-α on the cell surface. These results demonstrate that the TACE-binding peptide is an effective antagonist of TACE.

  5. A polystyrene binding target-unrelated peptide isolated in the screening of phage display library.

    Science.gov (United States)

    Bakhshinejad, Babak; Sadeghizadeh, Majid

    2016-11-01

    Phage display is a powerful methodology for the identification of peptide ligands binding to any desired target. However, the selection of target-unrelated peptides (TUPs) appears as a huge problem in the screening of phage display libraries through biopanning. The phage-displayed peptide TLHPAAD has been isolated both in our laboratory and by another reserach group on completely different screening targets prompting us to hypothesize that it may be a potential TUP. In the current study, we analyzed the binding characteristics and propagation rate of phage clone displaying TLHPAAD peptide (SW-TUP clone). The results of ELISA experiment and phage recovery assay provided strong support for the notion that SW-TUP phage binds to polystyrene with a significantly higher affinity than control phage clones. Furthermore, this polystyrene binding was demonstrated to occur in a concentration- and pH-dependent mode. Characterization of the propagation profile of phage clones within a specified time course revealed no statistically significant difference between the amplification rate of SW-TUP and control phages. Our findings lead us to the conclusion that SW-TUP phage clone with the displayed peptide TLHPAAD is not a true target binder and its selection in biopanning experiments results from its bidning affinity to the polystyrene surface of the solid phase.

  6. Phage displaying epitope of Candida albicans HSP90 and serodiagnosis

    Institute of Scientific and Technical Information of China (English)

    杨琼; 王丽; 卢大宁; 邢沈阳; 尹东; 朱筱娟

    2004-01-01

    @@ Recently, the frequent use of immunosuppressants and chemotherapeutic drugs for cancers has caused an increase in the frequency of life-threatening systemic candidiasis.1 Studies by Matthews et al2 indicated HSP90 fragments are major targets for the immune system in infection due to C. albicans, and anti-epitope LKVIRK of HSP90 antibody is a serological marker for diagnosis of invasive candidiasis. Cloning and sequencing HSP90 antigen revealed that the linear epitope LKVIRK, localized near the C-terminus of the 47 kDa protein which circulates in the sera of patients with invasive candidiasis, as a heat-stable breakdown product of large more heat-labile antigen HSP90.2 In this study, epitope LKVIRK was displayed on the surface of phage fd to develop a new serological test for systemic candidiasis.

  7. An efficient method for isolating antibody fragments against small peptides by antibody phage display

    DEFF Research Database (Denmark)

    Duan, Zhi; Siegumfeldt, Henrik

    2010-01-01

    We generated monoclonal scFv (single chain variable fragment) antibodies from an antibody phage display library towards three small synthetic peptides derived from the sequence of s1-casein. Key difficulties for selection of scFv-phages against small peptides were addressed. Small peptides do....... The scFvs were sequenced and characterized, and specificity was characterized by ELISA. The methods developed in this study are universally applicable for antibody phage display to efficiently produce antibody fragments against small peptides....

  8. Automated panning and screening procedure on microplates for antibody generation from phage display libraries.

    Science.gov (United States)

    Turunen, Laura; Takkinen, Kristiina; Söderlund, Hans; Pulli, Timo

    2009-03-01

    Antibody phage display technology is well established and widely used for selecting specific antibodies against desired targets. Using conventional manual methods, it is laborious to perform multiple selections with different antigens simultaneously. Furthermore, manual screening of the positive clones requires much effort. The authors describe optimized and automated procedures of these processes using a magnetic bead processor for the selection and a robotic station for the screening step. Both steps are performed in a 96-well microplate format. In addition, adopting the antibody phage display technology to automated platform polyethylene glycol precipitation of the enriched phage pool was unnecessary. For screening, an enzyme-linked immunosorbent assay protocol suitable for a robotic station was developed. This system was set up using human gamma-globulin as a model antigen to select antibodies from a VTT naive human single-chain antibody (scFv) library. In total, 161 gamma-globulin-selected clones were screened, and according to fingerprinting analysis, 9 of the 13 analyzed clones were different. The system was further tested using testosterone bovine serum albumin (BSA) and beta-estradiol-BSA as antigens with the same library. In total, 1536 clones were screened from 4 rounds of selection with both antigens, and 29 different testosterone-BSA and 23 beta-estradiol-BSA binding clones were found and verified by sequencing. This automated antibody phage display procedure increases the throughput of generating wide panels of target-binding antibody candidates and allows the selection and screening of antibodies against several different targets in parallel with high efficiency.

  9. Screening of Antigen Mimotopes of Rabbit Hemorrhagic Disease Virus by Phage Display Technology%应用噬菌体展示技术筛选兔出血症病毒抗原模拟表位

    Institute of Scientific and Technical Information of China (English)

    杨廷亚; 王芳; 姜平; 胡波; 范志宇; 魏后军; 薛家宾

    2012-01-01

    以抗兔出血症病毒(RHDV)的单克隆抗体A3c作为靶物质,应用噬菌体展示技术筛选RHDV抗原表位.将纯化的单抗A3c包被固相载体,经3轮亲和筛选后,挑取25株噬菌体单克隆并扩增,用EIISA测定后,提取阳性克隆单链DNA并测序,用阳性噬菌体克隆免疫小鼠制备高免血清,检测筛选抗原表位的免疫原性.结果表明:3轮亲和筛选后,特异性噬菌体克隆得到了有效富集,25株噬菌体单克隆中有19株为阳性克隆;测序结果表明,获得了与抗原高度同源的序列GTDDMDPGTTAA,即抗原的模拟表位,其中,氨基酸基序DXXDP为表位中的核心氨基酸;制备的小鼠高免血清与抗原具有较好的反应性,阳性噬菌体克隆与兔RHDV高免血清也具有较好的反应性.因此,该表位具有良好的免疫原性和反应原性.该研究为RHDV抗原表位的研究和新型疫苗的探索积累了资料.%The monoclonal antibody (McAb) A3c against rabbit hemorrhagic disease virus had been used as solid-phase selective molecule to screen the epitope of RHDV by phage display technology. McAb A3c was coated on the solid-phase and then three rounds of biopanning were carried out; 25 phages were selected and amplified to be identified by ELISA, and the positives were sequenced; the serum of mice immunized three times was prepared to determine the immunogenic-ity of the epitope. The results showed that the specific phages were enriched effectively after three rounds of biopanning; 19 of 25 phages were positive. The sequence analysis of the positive clones showed that highly homogenous sequence (GTDDMDPGTTAA) comparing to antigen was got, which is the antigen mimotopes of RHDV. The sequence DXXDP was the core amino acids in the epitope. In addition, the serum of mice reacted with antigen well and so did positive phages with hyper-immune serum of rabbit against RHDV, which indicated that the epo'tipe had good immunogenicity and reactionogenicity. This study provide

  10. Trinucleotide cassettes increase diversity of T7 phage-displayed peptide library

    Directory of Open Access Journals (Sweden)

    McMahon James B

    2007-10-01

    Full Text Available Abstract Background Amino acid sequence diversity is introduced into a phage-displayed peptide library by randomizing library oligonucleotide DNA. We recently evaluated the diversity of peptide libraries displayed on T7 lytic phage and M13 filamentous phage and showed that T7 phage can display a more diverse amino acid sequence repertoire due to differing processes of viral morphogenesis. Methods In this study, we evaluated and compared the diversity of a 12-mer T7 phage-displayed peptide library randomized using codon-corrected trinucleotide cassettes with a T7 and an M13 12-mer phage-displayed peptide library constructed using the degenerate codon randomization method. Results We herein demonstrate that the combination of trinucleotide cassette amino acid codon randomization and T7 phage display construction methods resulted in a significant enhancement to the functional diversity of a 12-mer peptide library. This novel library exhibited superior amino acid uniformity and order-of-magnitude increases in amino acid sequence diversity as compared to degenerate codon randomized peptide libraries. Comparative analyses of the biophysical characteristics of the 12-mer peptide libraries revealed the trinucleotide cassette-randomized library to be a unique resource. Conclusion The combination of T7 phage display and trinucleotide cassette randomization resulted in a novel resource for the potential isolation of binding peptides for new and previously studied molecular targets.

  11. Purification of phage display-modified bacteriophage T4 by affinity chromatography

    Directory of Open Access Journals (Sweden)

    Figura Grzegorz

    2011-05-01

    Full Text Available Abstract Background Affinity chromatography is one of the most efficient protein purification strategies. This technique comprises a one-step procedure with a purification level in the order of several thousand-fold, adaptable for various proteins, differentiated in their size, shape, charge, and other properties. The aim of this work was to verify the possibility of applying affinity chromatography in bacteriophage purification, with the perspective of therapeutic purposes. T4 is a large, icosahedral phage that may serve as an efficient display platform for foreign peptides or proteins. Here we propose a new method of T4 phage purification by affinity chromatography after its modification with affinity tags (GST and Histag by in vivo phage display. As any permanent introduction of extraneous DNA into a phage genome is strongly unfavourable for medical purposes, integration of foreign motifs with the phage genome was not applied. The phage was propagated in bacteria expressing fusions of the phage protein Hoc with affinity tags from bacterial plasmids, independently from the phage expression system. Results Elution profiles of phages modified with the specific affinity motifs (compared to non-specific phages document their binding to the affinity resins and effective elution with standard competitive agents. Non-specific binding was also observed, but was 102-105 times weaker than the specific one. GST-modified bacteriophages were also effectively released from glutathione Sepharose by proteolytic cleavage. The possibility of proteolytic release was designed at the stage of expression vector construction. Decrease in LPS content in phage preparations was dependent on the washing intensity; intensive washing resulted in preparations of 11-40 EU/ml. Conclusions Affinity tags can be successfully incorporated into the T4 phage capsid by the in vivo phage display technique and they strongly elevate bacteriophage affinity to a specific resin. Affinity

  12. A LIVER-TUMOR ADHESION PEPTIDE FROM RANDOM PHAGE DISPLAY LIBRARY

    Institute of Scientific and Technical Information of China (English)

    WU Miao; DU Bing; WANG Lei; ZHOU Zhong-liang; QIAN Min

    2006-01-01

    Objective: To identify and localize the synthesized targeting peptide A54 to liver cancer cell line BEL-7402 in vivo and in vitro for confirming the potential clinical application of peptide A54 in hepatocarcinoma targeting therapy. Methods: Phage A54 was confirmed by ELISA. Biotin and FAM labeled A54 peptides were identified and localized by means of immunohistochemistry and immunocytochemistry. Results: A54 peptide could target the liver-tumor tissue in vivo and adhere to several liver-tumor cells in vitro. FAM-labeled A54 peptides were localized on the membrane surface of liver-tumor cells. Conclusion: Synthesized A54 peptide obtained from in vivo phage display technology still kept special ability to adhere liver-tumor cell in vivo and in vitro. The A54 peptide could be a candidate carrier for hepatocarcinoma targeting therapy.

  13. Intra-domain phage display (ID-PhD of peptides and protein mini-domains censored from canonical pIII phage display

    Directory of Open Access Journals (Sweden)

    Katrina F Tjhung

    2015-04-01

    Full Text Available In this paper, we describe multivalent display of peptide and protein sequences typically censored from traditional N-terminal display on protein pIII of filamentous bacteriophage M13. Using site-directed mutagenesis of commercially available M13KE phage cloning vector, we introduced sites that permit efficient cloning using restriction enzymes between domains N1 and N2 of the pIII protein. As infectivity of phage is directly linked to the integrity of the connection between N1 and N2 domains, intra-domain phage display (ID-PhD allows for simple quality control of the display and the natural variations in the displayed sequences. Additionally, direct linkage to phage propagation allows efficient monitoring of sequence cleavage, providing a convenient system for selection and evolution of protease-susceptible or protease-resistant sequences. As an example of the benefits of such an ID-PhD system, we displayed a charged FLAG sequence, which is known to be post-translationally excised from pIII when displayed on the N-terminus. ID-PhD of FLAG exhibited sub-nanomolar apparent Kd suggesting multivalent nature of the display. A TEV-protease recognition sequence (TEVrs co-expressed in tandem with FLAG, allowed us to demonstrate that 99.9997% of the phage displayed the FLAG-TEVrs tandem and can be recognized and cleaved by TEV-protease. The residual 0.0003% consisted of phage clones that have excised the insert from their genome. ID-PhD is also amenable to display of protein mini-domains, such as the 33-residue minimized Z-domain of protein A. We show that it is thus possible to use ID-PhD for multivalent display and selection of mini-domain proteins (Affibodies, scFv, etc..

  14. Engineering RNA phage MS2 virus-like particles for peptide display

    Science.gov (United States)

    Jordan, Sheldon Keith

    Phage display is a powerful and versatile technology that enables the selection of novel binding functions from large populations of randomly generated peptide sequences. Random sequences are genetically fused to a viral structural protein to produce complex peptide libraries. From a sufficiently complex library, phage bearing peptides with practically any desired binding activity can be physically isolated by affinity selection, and, since each particle carries in its genome the genetic information for its own replication, the selectants can be amplified by infection of bacteria. For certain applications however, existing phage display platforms have limitations. One such area is in the field of vaccine development, where the goal is to identify relevant epitopes by affinity-selection against an antibody target, and then to utilize them as immunogens to elicit a desired antibody response. Today, affinity selection is usually conducted using display on filamentous phages like M13. This technology provides an efficient means for epitope identification, but, because filamentous phages do not display peptides in the high-density, multivalent arrays the immune system prefers to recognize, they generally make poor immunogens and are typically useless as vaccines. This makes it necessary to confer immunogenicity by conjugating synthetic versions of the peptides to more immunogenic carriers. Unfortunately, when introduced into these new structural environments, the epitopes often fail to elicit relevant antibody responses. Thus, it would be advantageous to combine the epitope selection and immunogen functions into a single platform where the structural constraints present during affinity selection can be preserved during immunization. This dissertation describes efforts to develop a peptide display system based on the virus-like particles (VLPs) of bacteriophage MS2. Phage display technologies rely on (1) the identification of a site in a viral structural protein that is

  15. Isolation of Human Antibodies Against Hepatitis E From Phage Display Library by Metal Affinity Chromatography

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective To isolate human antibodies against hepatitis E virus from phage display library by a new method of panning phage antibody library based on immobilized metal affinity chromatography (IMAC). Methods Phage antibody library was allowed to mix with hex-His tagged expressed HEV specific antigen, NE2, in solution for adequate binding before affinity resin for hex-His was added. The non-specific phage antibodies were removed by extensive washing and the specific bound phage antibodies could then be eluted to infect TG1 or repeat the binding process for subsequent rounds of purification. The specificity of the selected human antibodies were tested by antigen competitive ELISA, human sera blocking ELISA, scFv expression, and sequence analysis. Results His-NE2 specific recombinant phages were successfully enriched after panning procedure. Two individual phage clones, 126 and 138, showed 50% inhibition in NE2 antigen competition ELISA and obvious blocking effect by HEV positive serum in blocking ELISA. Soluble scFv of 126, 138 bound to NE2 specifically. Conclusion Two specific human phage antibodies against hepatitis E virus (HEV) from phage display library were isolated by immobilized metal affinity chromatography. The immobilized metal affinity chromatography applied to phage antibody selection was a helpful supplement to the selection in solution.

  16. Injected phage-displayed-VP28 vaccine reduces shrimp Litopenaeus vannamei mortality by white spot syndrome virus infection.

    Science.gov (United States)

    Solís-Lucero, G; Manoutcharian, K; Hernández-López, J; Ascencio, F

    2016-08-01

    White spot syndrome virus (WSSV) is the most important viral pathogen for the global shrimp industry causing mass mortalities with huge economic losses. Recombinant phages are capable of expressing foreign peptides on viral coat surface and act as antigenic peptide carriers bearing a phage-displayed vaccine. In this study, the full-length VP28 protein of WSSV, widely known as potential vaccine against infection in shrimp, was successfully cloned and expressed on M13 filamentous phage. The functionality and efficacy of this vaccine immunogen was demonstrated through immunoassay and in vivo challenge studies. In ELISA assay phage-displayed VP28 was bind to Litopenaeus vannamei immobilized hemocyte in contrast to wild-type M13 phage. Shrimps were injected with 2 × 10(10) cfu animal(-1) single dose of VP28-M13 and M13 once and 48 h later intramuscularly challenged with WSSV to test the efficacy of the vaccine against the infection. All dead challenged shrimps were PCR WSSV-positive. The accumulative mortality of the vaccinated and challenged shrimp groups was significantly lower (36.67%) than the unvaccinated group (66.67%). Individual phenoloxidase and superoxide dismutase activity was assayed on 8 and 48 h post-vaccination. No significant difference was found in those immunological parameters among groups at any sampled time evaluated. For the first time, phage display technology was used to express a recombinant vaccine for shrimp. The highest percentage of relative survival in vaccinated shrimp (RPS = 44.99%) suggest that the recombinant phage can be used successfully to display and deliver VP28 for farmed marine crustaceans.

  17. Silent Encoding of Chemical Post-Translational Modifications in Phage-Displayed Libraries.

    Science.gov (United States)

    Tjhung, Katrina F; Kitov, Pavel I; Ng, Simon; Kitova, Elena N; Deng, Lu; Klassen, John S; Derda, Ratmir

    2016-01-13

    In vitro selection of chemically modified peptide libraries presented on phage, while a powerful technology, is limited to one chemical post-translational modification (cPTM) per library. We use unique combinations of redundant codons to encode cPTMs with "silent barcodes" to trace multiple modifications within a mixed modified library. As a proof of concept, we produced phage-displayed peptide libraries Ser-[X]4-Gly-Gly-Gly, with Gly and Ser encoded using unique combinations of codons (TCA-[X]4-GGAGGAGGA, AGT-[X]4-GGTGGTGGT, etc., where [X]4 denotes a random NNK library). After separate chemical modification and pooling, mixed-modified libraries can be panned and deep-sequenced to identify the enriched peptide sequence and the accompanying cPTM simultaneously. We panned libraries bearing combinations of modifications (sulfonamide, biotin, mannose) against matched targets (carbonic anhydrase, streptavidin, concanavalin A) to identify desired ligands. Synthesis and validation of sequences identified by deep sequencing revealed that specific cPTMs are significantly enriched in panning against the specific targets. Panning on carbonic anhydrase yielded a potent ligand, sulfonamide-WIVP, with Kd = 6.7 ± 2.1 nM, a 20-fold improvement compared with the control ligand sulfonamide-GGGG. Silent encoding of multiple cPTMs can be readily incorporated into other in vitro display technologies such as bacteriophage T7 or mRNA display.

  18. Multifunctional g3p-peptide tag for current phage display systems.

    Science.gov (United States)

    Beckmann, C; Haase, B; Timmis, K N; Tesar, M

    1998-03-15

    . The high sensitivity of the antibody for the peptide tag was reflected in the antibody affinity constant K(D) of 6.80 x 10(-10) M, which was determined by real time biomolecular interaction analysis (BIA) based on surface plasmon resonance (SPR) [Karlsson, R., Fält, A., 1997. Experimental design for kinetic analysis of protein-protein interactions with surface plasmon resonance biosensors. J. Immunol. Methods 200, 121-133]. Finally, recombinant proteins in E. coli periplasmic extracts could be purified in a single step by affinity purification using immobilized mAb 10C3. These studies demonstrated that the new peptide-tag and its corresponding mAb represents a versatile tool for the detection of recombinant proteins selected by phage display technology.

  19. Development of a renal collecting duct homing peptide using phage display

    DEFF Research Database (Denmark)

    Svenningsen, Per; Peti-Peterdi, Janos

    Homing peptides are useful for in vivo labeling and nonviral gene transfer to selective tissues and cell types. The aim of this project was to develop a renal collecting duct homing peptide. Using phage display, we identified a phage expressing a cyclic 7 amino acid peptide, which was internalize...

  20. Next-generation phage display: integrating and comparing available molecular tools to enable cost-effective high-throughput analysis.

    Directory of Open Access Journals (Sweden)

    Emmanuel Dias-Neto

    Full Text Available BACKGROUND: Combinatorial phage display has been used in the last 20 years in the identification of protein-ligands and protein-protein interactions, uncovering relevant molecular recognition events. Rate-limiting steps of combinatorial phage display library selection are (i the counting of transducing units and (ii the sequencing of the encoded displayed ligands. Here, we adapted emerging genomic technologies to minimize such challenges. METHODOLOGY/PRINCIPAL FINDINGS: We gained efficiency by applying in tandem real-time PCR for rapid quantification to enable bacteria-free phage display library screening, and added phage DNA next-generation sequencing for large-scale ligand analysis, reporting a fully integrated set of high-throughput quantitative and analytical tools. The approach is far less labor-intensive and allows rigorous quantification; for medical applications, including selections in patients, it also represents an advance for quantitative distribution analysis and ligand identification of hundreds of thousands of targeted particles from patient-derived biopsy or autopsy in a longer timeframe post library administration. Additional advantages over current methods include increased sensitivity, less variability, enhanced linearity, scalability, and accuracy at much lower cost. Sequences obtained by qPhage plus pyrosequencing were similar to a dataset produced from conventional Sanger-sequenced transducing-units (TU, with no biases due to GC content, codon usage, and amino acid or peptide frequency. These tools allow phage display selection and ligand analysis at >1,000-fold faster rate, and reduce costs approximately 250-fold for generating 10(6 ligand sequences. CONCLUSIONS/SIGNIFICANCE: Our analyses demonstrates that whereas this approach correlates with the traditional colony-counting, it is also capable of a much larger sampling, allowing a faster, less expensive, more accurate and consistent analysis of phage enrichment. Overall

  1. Design and Screening of M13 Phage Display cDNA Libraries

    Directory of Open Access Journals (Sweden)

    Yuliya Georgieva

    2011-02-01

    Full Text Available The last decade has seen a steady increase in screening of cDNA expression product libraries displayed on the surface of filamentous bacteriophage. At the same time, the range of applications extended from the identification of novel allergens over disease markers to protein-protein interaction studies. However, the generation and selection of cDNA phage display libraries is subjected to intrinsic biological limitations due to their complex nature and heterogeneity, as well as technical difficulties regarding protein presentation on the phage surface. Here, we review the latest developments in this field, discuss a number of strategies and improvements anticipated to overcome these challenges making cDNA and open reading frame (ORF libraries more readily accessible for phage display. Furthermore, future trends combining phage display with next generation sequencing (NGS will be presented.

  2. Enhanced pulmonary absorption of a macromolecule through coupling to a sequence-specific phage display-derived peptide.

    Science.gov (United States)

    Morris, Christopher J; Smith, Mathew W; Griffiths, Peter C; McKeown, Neil B; Gumbleton, Mark

    2011-04-10

    With the aim of identifying a peptide sequence that promotes pulmonary epithelial transport of macromolecule cargo we used a stringent peptide-phage display library screening protocol against rat lung alveolar epithelial primary cell cultures. We identified a peptide-phage clone (LTP-1) displaying the disulphide-constrained 7-mer peptide sequence, C-TSGTHPR-C, that showed significant pulmonary epithelial translocation across highly restrictive polarised cell monolayers. Cell biological data supported a differential alveolar epithelial cell interaction of the LTP-1 peptide-phage clone and the corresponding free synthetic LTP-1 peptide. Delivering select phage-clones to the intact pulmonary barrier of an isolated perfused rat lung (IPRL) resulted in 8.7% of lung deposited LTP-1 peptide-phage clone transported from the IPRL airways to the vasculature compared (pprobes. This study shows proof-of principle that array technologies can be effectively exploited to identify peptides mediating enhanced transmucosal delivery of macromolecule therapeutics across an intact organ.

  3. Isolation by phage display and characterization of a singlechain antibody specific for O6-methyldeoxyguanosine

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    New approaches of making single chain Fv antibodies against O6-methyl-2′-deoxyguanosine (O6MdG) have been demonstrated by using the phage antibody display system. Using O6MdG as an antigen, 21 positive clones were identified by ELISA from this library, one of which, desig-nated H3, specifically binds to O6MdG with high affinity. The H3 scFv antibody has an affinity constant (Kaff) of 5.94×1011(mol/L)-1. H3 scFv has been successfully used to detect O6MdG in DNA hydrolyses from yeast or E. coli cells treated with a DNA methylating agent. To our knowledge, this is the first report of the selection of a specific scFv against DNA adducts. The results demonstrate the potential applications of the phage display technology for the detection of DNA lesions caused by mutagens and carcinogens.

  4. Selection of binding targets in parasites using phage-display and aptamer libraries in vivo and in vitro

    Directory of Open Access Journals (Sweden)

    Renata Rosito Tonelli

    2013-01-01

    Full Text Available Parasite infections are largely dependent on interactions between pathogen and different host cell populations to guarantee a successful infectious process. This is particularly true for obligatory intracellular parasites as Plasmodium, Toxoplasma, Leishmania, to name a few. Adhesion to and entry into the cell are essential steps requiring specific parasite and host cell molecules. The large amount of possible involved molecules poses additional difficulties for their identification by the classical biochemical approaches. In this respect, the search for alternative techniques should be pursued. Among them two powerful methodologies can be employed, both relying upon the construction of highly diverse combinatorial libraries of peptides or oligonucleotides that randomly bind with high affinity to targets on the cell surface and are selectively displaced by putative ligands. These are, respectively, the peptide-based phage display and the oligonucleotide-based aptamer techniques.The phage display technique has been extensively employed for the identification of novel ligands in vitro and in vivo in different areas such as cancer, vaccine development and epitope mapping. Particularly, phage display has been employed in the investigation of pathogen-host interactions. Although this methodology has been used for some parasites with encouraging results, in trypanosomatids its use is, as yet, scanty. RNA and DNA aptamers, developed by the SELEX process (Systematic Evolution of Ligands by Exponential Enrichment, were described over two decades ago and since then contributed to a large number of structured nucleic acids for diagnostic or therapeutic purposes or for the understanding of the cell biology. Similarly to the phage display technique scarce use of the SELEX process has been used in the probing of parasite-host interaction.In this review, an overall survey on the use of both phage display and aptamer technologies in different pathogenic

  5. Graphical analysis of flow cytometer data for characterizing controlled fluorescent protein display on λ phage.

    Science.gov (United States)

    Sokolenko, Stanislav; Nicastro, Jessica; Slavcev, Roderick; Aucoin, Marc G

    2012-12-01

    As native virus particles typically cannot be resolved using a flow cytometer, the general practice is to use fluorescent dyes to label the particles. In this work, an attempt was made to use a common commercial flow cytometer to characterize a phage display strategy that allows for controlled levels of protein display, in this case, eGFP. To achieve this characterization, a number of data processing steps were needed to ensure that the observed phenomena were indeed capturing differences in the phages produced. Phage display of eGFP resulted in altered side scatter and fluorescence profile, and sub-populations could be identified within what would otherwise be considered uniform populations. Surprisingly, this study has found that side scatter may be used in the future to characterize the display of nonfluorescent proteins.

  6. Handbook of Visual Display Technology

    CERN Document Server

    Cranton, Wayne; Fihn, Mark

    2012-01-01

    The Handbook of Visual Display Technology is a unique work offering a comprehensive description of the science, technology, economic and human interface factors associated with the displays industry. An invaluable compilation of information, the Handbook will serve as a single reference source with expert contributions from over 150 international display professionals and academic researchers. All classes of display device are covered including LCDs, reflective displays, flexible solutions and emissive devices such as OLEDs and plasma displays, with discussion of established principles, emergent technologies, and particular areas of application. The wide-ranging content also encompasses the fundamental science of light and vision, image manipulation, core materials and processing techniques, display driving and metrology.

  7. Phage display selects for amylases with improved low pH starch-binding

    NARCIS (Netherlands)

    Verhaert, RMD; Beekwilder, J; Olsthoorn, R; Quax, WJ; Duin, Jan van

    2002-01-01

    Directed evolution of secreted industrial enzymes is hampered by the lack of powerful selection techniques. We have explored surface display to select for enzyme variants with improved binding performance on complex polymeric substrates. By a combination of saturation mutagenesis and phage display w

  8. Phage display selects for amylases with improved low pH starch-binding

    NARCIS (Netherlands)

    Verhaert, RMD; Beekwilder, J; Olsthoorn, R; Quax, WJ; Duin, Jan van

    2002-01-01

    Directed evolution of secreted industrial enzymes is hampered by the lack of powerful selection techniques. We have explored surface display to select for enzyme variants with improved binding performance on complex polymeric substrates. By a combination of saturation mutagenesis and phage display

  9. Triosephosphate isomerase of Taenia solium (TTPI): phage display and antibodies as tools for finding target regions to inhibit catalytic activity.

    Science.gov (United States)

    Sanabria-Ayala, Víctor; Belmont, Iaraset; Abraham, Landa

    2015-01-01

    Previous studies demonstrated that antibodies against triosephosphate isomerase of Taenia solium (TTPI) can alter its enzymatic catalysis. In the present study, we used antibodies produced against the NH2-terminal region of TTPI (1/3NH2TTPI) and the phage display technology to find target regions to inhibit TTPI activity. As a first step, we obtained polyclonal antibodies against non-conserved regions from the 1/3NH2TTPI, which had an inhibitory effect of about 74 % on catalytic activity. Afterward, they were used to screen a library of phage-displayed dodecapeptides; as a result, 41 phage mimotope clones were isolated and grouped according to their amino acid sequence, finding the consensus A1 (VPTXPI), A2 (VPTXXI), B (LTPGQ), and D (DPLPR). Antibodies against selected phage mimotope clones were obtained by rabbit's immunization; these ones clearly recognized TTPI by both Western blot and ELISA. However, only the mimotope PDTS16 (DSVTPTSVMAVA) clone, which belongs to the VPTXXI consensus, raised antibodies capable of inhibiting the TTPI catalytic activity in 45 %. Anti-PDTS16 antibodies were confronted to several synthetic peptides that encompass the 1/3NH2TTPI, and they only recognized three, which share the motif FDTLQK belonging to the helix-α1 in TTPI. This suggests that this motif is the main part of the epitope recognized by anti-PDTS16 antibodies and revealed its importance for TTPI catalysis.

  10. Identification of nose-to-brain homing peptide through phage display.

    Science.gov (United States)

    Wan, Xiao-Mei; Chen, Yong-Ping; Xu, Wen-Rui; Yang, Wen-Jun; Wen, Long-Ping

    2009-02-01

    Brain delivery of drug molecules through the nasal passage represents a viable approach for bypassing the blood-brain barrier (BBB) but remains a major challenge due to lack of efficient homing carriers. To screen for potential peptides with the ability to transport into the brain via the nasal passage, we applied a C7C phage peptide display library (Ph.D.-C7C) intra-nasally to anesthetized rats and recovered phage from the brain tissue 45 min after phage administration. After three rounds of panning, 10 positive phage clones were selected and sequenced. Clone7, which exhibited highest translocation efficiency, was chosen for further studies. After nasal administration, Clone7 entered the brain within 30 min and exhibited translocation efficiency about 50-fold higher than a random phage. A 11-amino acid synthetic peptide derived from the displayed sequence of Clone7 (ACTTPHAWLCG) efficiently inhibited the nasal-brain translocation of Clone7. Both phage recovery results and fluorescent microscopy images revealed the presence of many more Clone7 phage in the brain than in the liver, kidney and other internal organs after the nasal administration, suggesting that Clone7 bypassed the BBB and entered brain directly. Furthermore, both Clone7 and the ACTTPHAWLCG peptide were found to be heavily distributed along the olfactory nerve after the nasal administration, further suggesting a direct passage route into the brain via the olfactory region. These results demonstrated the feasibility of using the in vivo phage display approach for selecting peptides with the nose-to-brain homing capability and may have implications for the development of novel targeting carriers useful for brain delivery.

  11. Generation and selection of immunized Fab phage display library against human B cell lymphoma

    Institute of Scientific and Technical Information of China (English)

    Yongmei Shen; Xiaochun Yang; Ningzheng Dong; Xiaofang Xie; Xia Bai; Yizhen Shi

    2007-01-01

    The approval of using monoclonal antibodies as a targeted therapy in the management of patients with B cell lymphoma has led to new treatment options for this group of patients. Production of monoclonal antibodies by the traditional hybridoma technology is costly, and the resulting murine antibodies often have the disadvantage of triggering human anti-mouse antibody (HAMA) response. Therefore recombinant Fab antibodies generated by the phage display technology can be a suitable alternative in managing B cell lymphoma. In this study, we extracted total RNA from spleen cells of BALB/c mice immunized with human B lymphoma cells, and used RT-PCR to amplify cDNAs coding for the K light chains and Fd fragments of heavy chains. After appropriate restriction digests, these cDNA fragments were successively inserted into the phagemid vector pComb3H-SS to construct an immunized Fab phage display library. The diversity of the constructed library was approximately 1.94×107. Following five rounds of biopanning, soluble Fab antibodies were produced from positive clones identified by ELISA. From eight positive clones, FabC06, FabC21, FabC43 and FabC59 were selected for sequence analysis. At the level of amino acid sequences, the variable heavy domains (VH) and variable light domains (VL) were found to share 88-92% and 89-94% homology with sequences coded by the corresponding murine germline genes respectively. Furthermore, reactivity with membrane proteins of the B cell lymphoma was demonstrated by immunohistochemistry and western blotting. These immunized Fab antibodies may provide a valuable tool for further study of B cell lymphoma and could also contribute to the improvement of disease therapy.

  12. Targeting Phosphatidylserine on Apoptotic Cells with Phages and Peptides Selected from a Bacteriophage Display Library

    Directory of Open Access Journals (Sweden)

    Ruping Shao

    2007-11-01

    Full Text Available Phosphatidylserine (PS is a well-characterized biomarker for apoptosis. Ligands that bind to PS can be used for noninvasive imaging of therapy-induced cell death, particularly apoptosis. In this study, we screened a random 12-mer peptide phage library on liposomes prepared from PS. One clone displaying the peptide SVSVGMKPSPRP (designated as PS3-10 bound to PS approximately 4-fold better than its binding to phosphatidylcholine and 18-fold better than to bovine serum albumin in a solid-phase binding assay. In addition, the binding of the corresponding PS3-10 peptide to PS was significantly higher than that of a scrambled peptide. PS3-10 phages, but not a control 4-2-2 phage, bound to aged red blood cells that had PS exposed on their surface. Binding of PS3-10 phages and PS3-10 peptide to TRAIL-induced apoptotic DLD1 cells was 3.2 and 5.4 times higher than their binding to untreated viable cells, respectively. Significantly, immunohistochemical staining confirmed selective binding of PS3-10 phages to apoptotic cells. Our data suggest that panning of phage display libraries may allow the selection of suitable peptide ligands for apoptotic cells and that PS3-10 peptide may serve as a template for further development of molecular probes for in vitro and in vivo imaging of apoptosis.

  13. Identification of human embryonic progenitor cell targeting peptides using phage display.

    Directory of Open Access Journals (Sweden)

    Paola A Bignone

    Full Text Available Human pluripotent stem (hPS cells are capable of differentiation into derivatives of all three primary embryonic germ layers and can self-renew indefinitely. They therefore offer a potentially scalable source of replacement cells to treat a variety of degenerative diseases. The ability to reprogram adult cells to induced pluripotent stem (iPS cells has now enabled the possibility of patient-specific hPS cells as a source of cells for disease modeling, drug discovery, and potentially, cell replacement therapies. While reprogramming technology has dramatically increased the availability of normal and diseased hPS cell lines for basic research, a major bottleneck is the critical unmet need for more efficient methods of deriving well-defined cell populations from hPS cells. Phage display is a powerful method for selecting affinity ligands that could be used for identifying and potentially purifying a variety of cell types derived from hPS cells. However, identification of specific progenitor cell-binding peptides using phage display may be hindered by the large cellular heterogeneity present in differentiating hPS cell populations. We therefore tested the hypothesis that peptides selected for their ability to bind a clonal cell line derived from hPS cells would bind early progenitor cell types emerging from differentiating hPS cells. The human embryonic stem (hES cell-derived embryonic progenitor cell line, W10, was used and cell-targeting peptides were identified. Competition studies demonstrated specificity of peptide binding to the target cell surface. Efficient peptide targeted cell labeling was accomplished using multivalent peptide-quantum dot complexes as detected by fluorescence microscopy and flow cytometry. The cell-binding peptides were selective for differentiated hPS cells, had little or no binding on pluripotent cells, but preferential binding to certain embryonic progenitor cell lines and early endodermal hPS cell derivatives. Taken

  14. Peptide substrate identification for yeast Hsp40 Ydj1 by screening the phage display library

    Directory of Open Access Journals (Sweden)

    Li Jingzhi

    2004-01-01

    Full Text Available We have identified a peptide substrate for molecular chaperone Hsp40 Ydj1 by utilizing the combination of phage display library screening and isothemol titration calirimetry (ITC. The initial peptide substrate screening for Hsp40 Ydj1 has been carried out by utilizing a 7-mer phage display library. The peptide sequences from the bio-panning were synthesized and object to the direct affinity measurement for Hsp40 Ydj1 by isothemol titration calirimetry studies. The peptide which has the measurable affinity with Ydj1 shows enriched hydrophobic residues in the middle of the substrate fragment. The peptide substrate specificity for molecular chaperone Hsp40 has been analyzed.

  15. Targeting pancreatic islets with phage display assisted by laser pressure catapult microdissection.

    Science.gov (United States)

    Yao, Virginia J; Ozawa, Michael G; Trepel, Martin; Arap, Wadih; McDonald, Donald M; Pasqualini, Renata

    2005-02-01

    Heterogeneity of the microvasculature in different organs has been well documented by multiple methods including in vivo phage display. However, less is known about the diversity of blood vessels within functionally distinct regions of organs. Here, we combined in vivo phage display with laser pressure catapult microdissection to identify peptide ligands for vascular receptors in the islets of Langerhans in the murine pancreas. Protein database analyses of the peptides, CVSNPRWKC and CHVLWSTRC, showed sequence identity to two ephrin A-type ligand homologues, A2 and A4. Confocal microscopy confirmed that most immunoreactivity of CVSNPRWKC and CHVLWSTRC phage was associated with blood vessels in pancreatic islets. Antibodies recognizing EphA4, a receptor for ephrin-A ligands, were similarly associated with islet blood vessels. Importantly, binding of both islet-homing phage and anti-EphA4 antibody was strikingly increased in blood vessels of pancreatic islet tumors in RIP-Tag2 transgenic mice. These results indicate that endothelial cells of blood vessels in pancreatic islets preferentially express EphA4 receptors, and this expression is increased in tumors. Our findings show in vivo phage display and laser pressure catapult microdissection can be combined to reveal endothelial cell specialization within focal regions of the microvasculature.

  16. Corruption of phage display libraries by target-unrelated clones: diagnosis and countermeasures.

    Science.gov (United States)

    Thomas, William D; Golomb, Miriam; Smith, George P

    2010-12-15

    Phage display is used to discover peptides or proteins with a desired target property-most often, affinity for a target selector molecule. Libraries of phage clones displaying diverse surface peptides are subject to a selection process designed to enrich for the target behavior and subsequently propagated to restore phage numbers. A recurrent problem is enrichment of clones, called target-unrelated phages or peptides (TUPs), that lack the target behavior. Many TUPs are propagation related; they have mutations conferring a growth advantage and are enriched during the propagations accompanying selection. Unlike other filamentous phage libraries, fd-tet-based libraries are relatively resistant to propagation-related TUP corruption. Their minus-strand origin is disrupted by a large cassette that simultaneously confers resistance to tetracycline and imposes a rate-limiting growth defect that cannot be bypassed with simple mutations. Nonetheless, a new type of propagation-related TUP emerged in the output of in vivo selections from an fd-tet library. The founding clone had a complex rearrangement that restored the minus-strand origin while retaining tetracycline resistance. The rearrangement involved two recombination events, one with a contaminant having a wild-type minus-strand origin. The founder's infectivity advantage spread by simple recombination to clones displaying different peptides. We propose measures for minimizing TUP corruption.

  17. Corruption of phage-display libraries by target-unrelated clones: Diagnosis and countermeasures

    Science.gov (United States)

    Thomas, William D.; Golomb, Miriam; Smith, George P.

    2010-01-01

    Phage display is used to discover peptides or proteins with a desired target property—most often, affinity for a target selector molecule. Libraries of phage clones displaying diverse surface peptides are subject to a selection process designed to enrich for the target behavior, and subsequently propagated to restore phage numbers. A recurrent problem is enrichment of clones, called target-unrelated phage (TUPs), that lack the target behavior. Many TUPs are propagation-related; they have mutations conferring a growth advantage, and are enriched during the propagations accompanying selection. Unlike other filamentous phage libraries, fd-tet-based libraries are relatively resistant to propagation-related TUP corruption. Their minus strand origin is disrupted by a large cassette that simultaneously confers resistance to tetracycline and imposes a rate-limiting growth defect that cannot be bypassed with simple mutations. Nonetheless, a new type of propagation-related TUP emerged in the output of in vivo selections from an fd-tet library. The founding clone had a complex rearrangement that restored the minus strand origin while retaining tetracycline resistance. The rearrangement involved two recombination events, one with a contaminant having a wild-type minus strand origin. The founder’s infectivity advantage spread by simple recombination to clones displaying different peptides. We propose measures for minimizing TUP corruption. PMID:20692225

  18. An Electrochemiluminescence Immunosensor Based on Gold-Magnetic Nanoparticles and Phage Displayed Antibodies.

    Science.gov (United States)

    Mu, Xihui; Tong, Zhaoyang; Huang, Qibin; Liu, Bing; Liu, Zhiwei; Hao, Lanqun; Dong, Hua; Zhang, Jinping; Gao, Chuan

    2016-02-27

    Using the multiple advantages of the ultra-highly sensitive electrochemiluminescence (ECL) technique, Staphylococcus protein A (SPA) functionalized gold-magnetic nanoparticles and phage displayed antibodies, and using gold-magnetic nanoparticles coated with SPA and coupled with a polyclonal antibody (pcAb) as magnetic capturing probes, and Ru(bpy)₃(2+)-labeled phage displayed antibody as a specific luminescence probe, this study reports a new way to detect ricin with a highly sensitive and specific ECL immunosensor and amplify specific detection signals. The linear detection range of the sensor was 0.0001~200 µg/L, and the limit of detection (LOD) was 0.0001 µg/L, which is 2500-fold lower than that of the conventional ELISA technique. The gold-magnetic nanoparticles, SPA and Ru(bpy)₃(2+)-labeled phage displayed antibody displayed different amplifying effects in the ECL immunosensor and can decrease LOD 3-fold, 3-fold and 20-fold, respectively, compared with the ECL immunosensors without one of the three effects. The integrated amplifying effect can decrease the LOD 180-fold. The immunosensor integrates the unique advantages of SPA-coated gold-magnetic nanoparticles that improve the activity of the functionalized capturing probe, and the amplifying effect of the Ru(bpy)₃(2+)-labeled phage displayed antibodies, so it increases specificity, interference-resistance and decreases LOD. It is proven to be well suited for the analysis of trace amounts of ricin in various environmental samples with high recovery ratios and reproducibility.

  19. An Electrochemiluminescence Immunosensor Based on Gold-Magnetic Nanoparticles and Phage Displayed Antibodies

    Directory of Open Access Journals (Sweden)

    Xihui Mu

    2016-02-01

    Full Text Available Using the multiple advantages of the ultra-highly sensitive electrochemiluminescence (ECL technique, Staphylococcus protein A (SPA functionalized gold-magnetic nanoparticles and phage displayed antibodies, and using gold-magnetic nanoparticles coated with SPA and coupled with a polyclonal antibody (pcAb as magnetic capturing probes, and Ru(bpy32+-labeled phage displayed antibody as a specific luminescence probe, this study reports a new way to detect ricin with a highly sensitive and specific ECL immunosensor and amplify specific detection signals. The linear detection range of the sensor was 0.0001~200 µg/L, and the limit of detection (LOD was 0.0001 µg/L, which is 2500-fold lower than that of the conventional ELISA technique. The gold-magnetic nanoparticles, SPA and Ru(bpy32+-labeled phage displayed antibody displayed different amplifying effects in the ECL immunosensor and can decrease LOD 3-fold, 3-fold and 20-fold, respectively, compared with the ECL immunosensors without one of the three effects. The integrated amplifying effect can decrease the LOD 180-fold. The immunosensor integrates the unique advantages of SPA-coated gold-magnetic nanoparticles that improve the activity of the functionalized capturing probe, and the amplifying effect of the Ru(bpy32+-labeled phage displayed antibodies, so it increases specificity, interference-resistance and decreases LOD. It is proven to be well suited for the analysis of trace amounts of ricin in various environmental samples with high recovery ratios and reproducibility.

  20. Antibody engineering using phage display with a coiled-coil heterodimeric Fv antibody fragment.

    Directory of Open Access Journals (Sweden)

    Xinwei Wang

    Full Text Available A Fab-like antibody binding unit, ccFv, in which a pair of heterodimeric coiled-coil domains was fused to V(H and V(L for Fv stabilization, was constructed for an anti-VEGF antibody. The anti-VEGF ccFv showed the same binding affinity as scFv but significantly improved stability and phage display level. Furthermore, phage display libraries in the ccFv format were constructed for humanization and affinity maturation of the anti-VEGF antibody. A panel of V(H frameworks and V(H-CDR3 variants, with a significant improvement in affinity and expressibility in both E. coli and yeast systems, was isolated from the ccFv phage libraries. These results demonstrate the potential application of the ccFv antibody format in antibody engineering.

  1. Immunodiagnosis of Canine Visceral Leishmaniasis Using Mimotope Peptides Selected from Phage Displayed Combinatorial Libraries

    Directory of Open Access Journals (Sweden)

    Christina Monerat Toledo-Machado

    2015-01-01

    Full Text Available ELISA and RIFI are currently used for serodiagnosis of canine visceral leishmaniasis (CVL. The accuracy of these tests is controversial in endemic areas where canine infections by Trypanosoma cruzi may occur. We evaluated the usefulness of synthetic peptides that were selected through phage display technique in the serodiagnosis of CVL. Peptides were chosen based on their ability to bind to IgGs purified from infected dogs pooled sera. We selected three phage clones that reacted only with those IgGs. Peptides were synthesized, polymerized with glutaraldehyde, and used as antigens in ELISA assays. Each individual peptide or a mix of them was reactive with infected dogs serum. The assay was highly sensitive and specific when compared to soluble Leishmania antigen that showed cross-reactivity with anti-T. cruzi IgGs. Our results demonstrate that phage display technique is useful for selection of peptides that may represent valuable synthetic antigens for an improved serodiagnosis of CVL.

  2. Latest development of display technologies

    Science.gov (United States)

    Gao, Hong-Yue; Yao, Qiu-Xiang; Liu, Pan; Zheng, Zhi-Qiang; Liu, Ji-Cheng; Zheng, Hua-Dong; Zeng, Chao; Yu, Ying-Jie; Sun, Tao; Zeng, Zhen-Xiang

    2016-09-01

    In this review we will focus on recent progress in the field of two-dimensional (2D) and three-dimensional (3D) display technologies. We present the current display materials and their applications, including organic light-emitting diodes (OLEDs), flexible OLEDs quantum dot light emitting diodes (QLEDs), active-matrix organic light emitting diodes (AMOLEDs), electronic paper (E-paper), curved displays, stereoscopic 3D displays, volumetric 3D displays, light field 3D displays, and holographic 3D displays. Conventional 2D display devices, such as liquid crystal devices (LCDs) often result in ambiguity in high-dimensional data images because of lacking true depth information. This review thus provides a detailed description of 3D display technologies.

  3. Construction of Human ScFv Phage Display Library against Ovarian Tumor

    Institute of Scientific and Technical Information of China (English)

    XIA Jinsong; BI Hao; YAO Qin; QU Shen; ZONG Yiqiang

    2006-01-01

    In order to construct a single chain fragment variable (ScFv) phage display library against ovarian tumor, by using RT-PCR, the human heavy chain variable region genes (VH) and light chain variable region genes (VL) were amplified from lymphocytes of ovarian tumor patients and subsequently assembled into ScFv genes by SOE. The resulting ScFv genes were electrotransformed into E.coli TG1 and amplified with the co-infection of helper phage M13KO7 to obtain phage display library. The capacity and titer of the resulting library were detected. The phage antibody library with a capacity of approximately 3 × 109 cfu/μg was obtained. After amplification with helper phage, the titer of antibody library reached 5 × 1012 cfu/mL. Human ScFv library against ovarian tumor was constructed successfully, which laid a foundation for the screening of ovarian tumor specific ScFv for the radioimmunoimaging diagnosis of ovarian tumor.

  4. Selection of a breast cancer subpopulation-specific antibody using phage display on tissue sections

    DEFF Research Database (Denmark)

    Larsen, Simon Asbjørn; Meldgaard, Theresa; Fridriksdottir, Agla J

    2015-01-01

    of breast cancer may provide crucial knowledge for the development of individualized therapy. However, this process is challenged by the availability of biomarkers able to identify subpopulations specifically. Here, we demonstrate an approach for phage display selection of recombinant antibody fragments...

  5. A recombinant, fully human monoclonal antibody with antitumor activity constructed from phage-displayed antibody fragments

    NARCIS (Netherlands)

    Huls, GA; Heijnen, IAFM; Cuomo, ME; Koningsberger, JC; Boel, E; de Vries, ARV; Loyson, SAJ; Helfrich, W; Henegouwen, GPV; van Meijer, M; de Kruif, J; Logtenberg, T

    1999-01-01

    A single-chain Fv antibody fragment specific for the tumor-associated Ep-CAM molecule was isolated from a semisynthetic phage display library and converted into an intact, fully human IgG1 monoclonal antibody (huMab), The purified huMab had an affinity of 5 nM and effectively mediated tumor cell kil

  6. Chaperone-assisted thermostability engineering of a soluble T cell receptor using phage display

    DEFF Research Database (Denmark)

    Gunnarsen, Kristin S; Kristinsson, Solveig G; Justesen, Sune

    2013-01-01

    We here report a novel phage display selection strategy enabling fast and easy selection of thermostabilized proteins. The approach is illustrated with stabilization of an aggregation-prone soluble single chain T cell receptor (scTCR) characteristic of the murine MOPC315 myeloma model. Random...

  7. Isolation of llama antibody fragments for prevention of dandruff by phage display in shampoo

    NARCIS (Netherlands)

    Dolk, E.; Vaart, M. van der; Lutje Hulsik, D.; Vriend, G.; Haard, H. de; Spinelli, S.; Cambillau, C.; Frenken, L.; Verrips, T.

    2005-01-01

    As part of research exploring the feasibility of using antibody fragments to inhibit the growth of organisms implicated in dandruff, we isolated antibody fragments that bind to a cell surface protein of Malassezia furfur in the presence of shampoo. We found that phage display of llama single-domain

  8. Selection and characterization of a unique phage display-derived antibody against dermatan sulfate.

    NARCIS (Netherlands)

    Lensen, J.F.M.; Wijnhoven, T.J.M.; Kuik, L.H.; Versteeg, E.M.M.; Hafmans, T.G.M.; Rops, A.L.; Pavao, M.S.; Vlag, J. van der; Heuvel, L.P.W.J. van den; Berden, J.H.M.; Kuppevelt, A.H.M.S.M. van

    2006-01-01

    Dermatan sulfate (DS) is a member of the glycosaminoglycan (GAG) family and is primarily located in the extracellular matrix. Using a modified phage display procedure, we selected 2 different antibodies against DS of which one antibody, LKN1, was specific for DS. LKN1 was especially reactive with

  9. Phage-display libraries of murine and human antibody Fab fragments

    DEFF Research Database (Denmark)

    Engberg, J; Andersen, P S; Nielsen, L K

    1996-01-01

    We provide efficient and detailed procedures for construction, expression, and screening of comprehensive libraries of murine or human antibody Fab fragments displayed on the surface of filamentous phage. In addition, protocols for producing and using ultra-electrocompetent cells, for producing Fab...

  10. An efficient method for variable region assembly in the construction of scFv phage display libraries using independent strand amplification.

    Science.gov (United States)

    Sotelo, Pablo; Collazo, Noberto; Zuñiga, Roberto; Gutiérrez-González, Matías; Catalán, Diego; Ribeiro, Carolina Hager; Aguillón, Juan Carlos; Molina, María Carmen

    2012-01-01

    Phage display library technology is a common method to produce human antibodies. In this technique, the immunoglobulin variable regions are displayed in a bacteriophage in a way that each filamentous virus displays the product of a single antibody gene on its surface. From the collection of different phages, it is possible to isolate the virus that recognizes specific targets. The most common form in which to display antibody variable regions in the phage is the single chain variable fragment format (scFv), which requires assembly of the heavy and light immunoglobulin variable regions in a single gene. In this work, we describe a simple and efficient method for the assembly of immunoglobulin heavy and light chain variable regions in a scFv format. This procedure involves a two-step reaction: (1) DNA amplification to produce the single strand form of the heavy or light chain gene required for the fusion; and (2) mixture of both single strand products followed by an assembly reaction to construct a complete scFv gene. Using this method, we produced 6-fold more scFv encoding DNA than the commonly used splicing by overlap extension PCR (SOE-PCR) approach. The scFv gene produced by this method also proved to be efficient in generating a diverse scFv phage display library. From this scFv library, we obtained phages that bound several non-related antigens, including recombinant proteins and rotavirus particles.

  11. Improvement and efficient display of Bacillus thuringiensis toxins on M13 phages and ribosomes.

    Science.gov (United States)

    Pacheco, Sabino; Cantón, Emiliano; Zuñiga-Navarrete, Fernando; Pecorari, Frédéric; Bravo, Alejandra; Soberón, Mario

    2015-12-01

    Bacillus thuringiensis (Bt) produces insecticidal proteins that have been used worldwide in the control of insect-pests in crops and vectors of human diseases. However, different insect species are poorly controlled by the available Bt toxins or have evolved resistance to these toxins. Evolution of Bt toxicity could provide novel toxins to control insect pests. To this aim, efficient display systems to select toxins with increased binding to insect membranes or midgut proteins involved in toxicity are likely to be helpful. Here we describe two display systems, phage display and ribosome display, that allow the efficient display of two non-structurally related Bt toxins, Cry1Ac and Cyt1Aa. Improved display of Cry1Ac and Cyt1Aa on M13 phages was achieved by changing the commonly used peptide leader sequence of the coat pIII-fusion protein, that relies on the Sec translocation pathway, for a peptide leader sequence that relies on the signal recognition particle pathway (SRP) and by using a modified M13 helper phage (Phaberge) that has an amber mutation in its pIII genomic sequence and preferentially assembles using the pIII-fusion protein. Also, both Cry1Ac and Cyt1Aa were efficiently displayed on ribosomes, which could allow the construction of large libraries of variants. Furthermore, Cry1Ac or Cyt1Aa displayed on M13 phages or ribosomes were specifically selected from a mixture of both toxins depending on which antigen was immobilized for binding selection. These improved systems may allow the selection of Cry toxin variants with improved insecticidal activities that could counter insect resistances.

  12. Phage Display: Selecting Straws Instead of a Needle from a Haystack

    Directory of Open Access Journals (Sweden)

    Mojca Lunder

    2011-01-01

    Full Text Available An increasing number of peptides with specific binding affinity to various protein and even non-protein targets are being discovered from phage display libraries. The power of this method lies in its ability to efficiently and rapidly identify ligands with a desired target property from a large population of phage clones displaying diverse surface peptides. However, the search for the needle in the haystack does not always end successfully. False positive results may appear. Thus instead of specific binders phage with no actual affinity toward the target are recovered due to their propagation advantages or binding to other components of the screening system, such as the solid phase, capturing reagents, contaminants in the target sample or blocking agents, rather than the target. Biopanning experiments on different targets performed in our laboratory revealed some previously identified and many new target-unrelated peptide sequences, which have already been frequently described and published, but not yet recognized as target-unrelated. Distinguishing true binders from false positives is an important step toward phage display selections of greater integrity. This article thoroughly reviews and discusses already identified and new target-unrelated peptides and suggests strategies to avoid their isolation.

  13. Identification of hepatitis A virus mimotopes by phage display, antigenicity and immunogenicity.

    Science.gov (United States)

    Larralde, Osmany G; Martinez, Raiza; Camacho, Frank; Amin, Nevis; Aguilar, Alicia; Talavera, Arturo; Stott, David I; Perez, Ela M

    2007-03-01

    A phage-displayed peptide approach was used to identify ligands mimicking antigenic determinants of hepatitis A virus (HAV) for the first time. Bacteriophages displaying HAV mimotopes were isolated from a phage-display peptide library by affinity selection on serum antibodies from hepatitis A patients. Selected phage-peptides were screened for reactivity with sera from HAV infected patients and healthy controls. Four cloned peptides with different sequences were identified as mimotopes of HAV; three of them showed similarity in their amino acid sequences with at least one of the VP3 and VP1 antigenic proteins of HAV. One clone was recognised by 92% of the positive sera. The phagotopes competed effectively with HAV for absorption of anti-HAV-specific antibodies in human sera, as determined by ELISA. The four phage clones induced neutralising anti-HAV antibodies in immunised mice. These results demonstrate the potential of this method to elucidate the disease related epitopes of HAV and to use these mimotopes in diagnostic applications or in the development of a mimotope-based hepatitis A vaccine without the necessity of manipulation of the virus.

  14. Phage display-derived binders able to distinguish Listeria monocytogenes from other Listeria species.

    Directory of Open Access Journals (Sweden)

    Josephine Morton

    Full Text Available The objective of this study was to produce phage display-derived binders with the ability to distinguish Listeria monocytogenes from other Listeria spp., which may have potential utility to enhance detection of Listeria monocytogenes. To obtain binders with the desired binding specificity a series of surface and solution phage-display biopannings were performed. Initially, three rounds of surface biopanning against gamma-irradiated L. monocytogenes serovar 4b cells were performed followed by an additional surface biopanning round against L. monocytogenes 4b which included prior subtraction biopanning against gamma-irradiated L. innocua cells. In an attempt to further enhance binder specificity for L. monocytogenes 4b two rounds of solution biopanning were performed, both rounds included initial subtraction solution biopanning against L. innocua. Subsequent evaluations were performed on the phage clones by phage binding ELISA. All phage clones tested from the second round of solution biopanning had higher specificity for L. monocytogenes 4b than for L. innocua and three other foodborne pathogens (Salmonella spp., Escherichia coli and Campylobacter jejuni. Further evaluation with five other Listeria spp. revealed that one phage clone in particular, expressing peptide GRIADLPPLKPN, was highly specific for L. monocytogenes with at least 43-fold more binding capability to L. monocytogenes 4b than to any other Listeria sp. This proof-of-principle study demonstrates how a combination of surface, solution and subtractive biopanning was used to maximise binder specificity. L. monocytogenes-specific binders were obtained which could have potential application in novel detection tests for L. monocytogenes, benefiting both the food and medical industries.

  15. Phage display-derived binders able to distinguish Listeria monocytogenes from other Listeria species.

    Science.gov (United States)

    Morton, Josephine; Karoonuthaisiri, Nitsara; Charlermroj, Ratthaphol; Stewart, Linda D; Elliott, Christopher T; Grant, Irene R

    2013-01-01

    The objective of this study was to produce phage display-derived binders with the ability to distinguish Listeria monocytogenes from other Listeria spp., which may have potential utility to enhance detection of Listeria monocytogenes. To obtain binders with the desired binding specificity a series of surface and solution phage-display biopannings were performed. Initially, three rounds of surface biopanning against gamma-irradiated L. monocytogenes serovar 4b cells were performed followed by an additional surface biopanning round against L. monocytogenes 4b which included prior subtraction biopanning against gamma-irradiated L. innocua cells. In an attempt to further enhance binder specificity for L. monocytogenes 4b two rounds of solution biopanning were performed, both rounds included initial subtraction solution biopanning against L. innocua. Subsequent evaluations were performed on the phage clones by phage binding ELISA. All phage clones tested from the second round of solution biopanning had higher specificity for L. monocytogenes 4b than for L. innocua and three other foodborne pathogens (Salmonella spp., Escherichia coli and Campylobacter jejuni). Further evaluation with five other Listeria spp. revealed that one phage clone in particular, expressing peptide GRIADLPPLKPN, was highly specific for L. monocytogenes with at least 43-fold more binding capability to L. monocytogenes 4b than to any other Listeria sp. This proof-of-principle study demonstrates how a combination of surface, solution and subtractive biopanning was used to maximise binder specificity. L. monocytogenes-specific binders were obtained which could have potential application in novel detection tests for L. monocytogenes, benefiting both the food and medical industries.

  16. Identification of peptides that selectively bind to myoglobin by biopanning of phage displayed-peptide library.

    Science.gov (United States)

    Padmanaban, Guruprasath; Park, Hyekyung; Choi, Ji Suk; Cho, Yong-Woo; Kang, Woong Chol; Moon, Chan-Il; Kim, In-San; Lee, Byung-Heon

    2014-10-10

    Biopanning of phage displayed-peptide library was performed against myoglobin, a marker for the early assessment of acute myocardial infarction (AMI), to identify peptides that selectively bind to myoglobin. Using myoglobin-conjugated magnetic beads, phages that bound to myoglobin were collected and amplified for the next round of screening. A 148-fold enrichment of phage titer was observed after five rounds of screening relative to the first round. After phage binding ELISA, three phage clones were selected (3R1, 3R7 and 3R10) and the inserted peptides were chemically synthesized. The analysis of binding affinity showed that the 3R7 (CPSTLGASC) peptide had higher binding affinity (Kd=57 nM) than did the 3R1 (CNLSSSWIC) and 3R10 (CVPRLSAPC) peptide (Kd=125 nM and 293 nM, respectively). Cross binding activity to other proteins, such as bovine serum albumin, troponin I, and creatine kinase-MB, was minimal. In a peptide-antibody sandwich ELISA, the selected peptides efficiently captured myoglobin. Moreover, the concentrations of myoglobin in serum samples measured by a peptide-peptide sandwich assay were comparable to those measured by a commercial antibody-based kit. These results indicate that the identified peptides can be used for the detection of myoglobin and may be a cost effective alternative to antibodies.

  17. Screening of a specific peptide binding to esophageal squamous carcinoma cells from phage displayed peptide library.

    Science.gov (United States)

    Ma, Caixia; Li, Chunyan; Jiang, Dongliang; Gao, Xiaojie; Han, Juanjuan; Xu, Nan; Wu, Qiong; Nie, Guochao; Chen, Wei; Lin, Fenghuei; Hou, Yingchun

    2015-06-01

    To select a specifically binding peptide for imaging detection of human esophageal squamous cell carcinoma (ESCC), a phage-displayed 12-mer peptide library was used to screen the peptide that bind to ESCC cells specifically. After four rounds of bio-panning, the phage recovery rate gradually increased, and specific phage clones were effectively enriched. The 60 randomly selected phage clones were tested using cellular enzyme-linked immunosorbent assay (ELISA), and 41 phage clones were identified as positive clones with the over 2.10 ratio of absorbance higher than other clones, IRP and PBS controls. From the sequencing results of the positive clones, 14 peptide sequences were obtained and ESCP9 consensus sequence was identified as the peptide with best affinity to ESCC cells via competitive inhibition, fluorescence microscopy, and flow cytometry. The results indicate that the peptide ESCP9 can bind to ESCC cells specifically and sensitively, and it is a potential candidate to be developed as an useful molecule to the imaging detection and targeting therapy for ESCC.

  18. Whole genome phage display selects for proline-rich Boi polypeptides against Bem1p.

    Science.gov (United States)

    Hertveldt, Kirsten; Robben, Johan; Volckaert, Guido

    2006-08-01

    Interaction selection by biopanning from a fragmented yeast proteome displayed on filamentous phage particles was successful in identifying proline-rich fragments of Boi1p and Boi2p. These proteins bind to the second "src homology region 3'' (SH3) domain of Bem1p, a protein of Saccharomyces cerevisiae involved in bud formation. Target Bem1p was a doubly-tagged recombinant, Bem1([Asn142-Ile551]), which strongly interacts in ELISA with a C-terminal 75 amino acids polypeptide from Cdc24p exposed on phage. The whole yeast genomic display library contained approximately 7.7 x 10(7) independent clones of sheared S. cerevisiae genomic DNA fused to a truncated M13 gene III. This study corroborates the value of fragmented-proteome display to identify strong and direct interacting protein modules.

  19. Romulus and Remus, two phage isolates representing a distinct clade within the Twortlikevirus genus, display suitable properties for phage therapy applications.

    Science.gov (United States)

    Vandersteegen, Katrien; Kropinski, Andrew M; Nash, John H E; Noben, Jean-Paul; Hermans, Katleen; Lavigne, Rob

    2013-03-01

    The renewed interest in controlling Staphylococcus aureus infections using their natural enemies, bacteriophages, has led to the isolation of a limited number of virulent phages so far. These phages are all members of the Twortlikevirus, displaying little variance. We present two novel closely related (95.9% DNA homology) lytic myoviruses, Romulus and Remus, with double-stranded DNA (dsDNA) genomes of 131,333 bp and 134,643 bp, respectively. Despite their relatedness to Staphylococcus phages K, G1, ISP, and Twort and Listeria phages A511 and P100, Romulus and Remus can be proposed as isolates of a new species within the Twortlikevirus genus. A distinguishing feature for these phage genomes is the unique distribution of group I introns compared to that in other staphylococcal myoviruses. In addition, a hedgehog/intein domain was found within their DNA polymerase genes, and an insertion sequence-encoded transposase exhibits splicing behavior and produces a functional portal protein. From a phage therapy application perspective, Romulus and Remus infected approximately 70% of the tested S. aureus isolates and displayed promising lytic activity against these isolates. Furthermore, both phages showed a rapid initial adsorption and demonstrated biofilm-degrading capacity in a proof-of-concept experiment.

  20. Selection of trkB-binding peptides from a phage-displayed random peptide library

    Institute of Scientific and Technical Information of China (English)

    马仲才; 吴晓兰; 曹明媚; 潘卫; 朱分禄; 陈景山; 戚中田

    2003-01-01

    Brain-derived neurotrophic factor (BDNF) shows potential in the treatment of neurodegenerative diseases, but the therapeutic application of BDNF has been greatly limited because it is too large in molecular size to permeate blood-brain barrier. To develop low-molecular-weight BDNF-like peptides, we selected a phage-displayed random peptide library using trkB expressed on NIH 3T3 cells as target in the study. With the strategy of peptide library incubation with NIH 3T3 cells and competitive elution with 1 υg/mL of BDNF in the last round of selection, the specific phages able to bind to the natural conformation of trkB and antagonize BDNF binding to trkB were enriched effectively. Five trkB-binding peptides were obtained, in which a core sequence of CRA/TXφXXφXXC (X represents the random amino acids, φ represents T, L or I) was identified. The BDNF-like activity of these five peptides displayed on phages was not observed, though all of them antagonized the activity of BDNF in a dose-dependent manner. Similar results were obtained with the synthetic peptide of C1 clone, indicating that the 5 phage-derived peptides were trkB antagonists. These low-molecular-weight antagonists of trkB may be of potential application in the treatment of neuroblastoma and chronic pain. Meanwhile, the obtained core sequence also could be used as the base to construct the secondary phage-displayed peptide library for further development of small peptides mimicking BDNF activity.

  1. Epitope Mapping of Dengue-Virus-Enhancing Monoclonal-Antibody Using Phage Display Peptide Library

    Directory of Open Access Journals (Sweden)

    Chung-I Rai

    2008-01-01

    Full Text Available The Antibody-Dependent Enhancement (ADE hypothesis has been proposed to explain why more severe manifestations of Dengue Hemorrhagic Fever and Dengue Shock Syndrome (DHF/DSS occur predominantly during secondary infections of Dengue Virus (DV with different serotypes. However, the epitopes recognized by these enhancing antibodies are unclear. Recently, anti-pre-M monoclonal antibody (mAb 70-21, which recognized all DV serotypes without neutralizing activity, were generated and demonstrated as an enhancing antibody for DV infection. In the present study, the epitope recognized by mAb 70-21 was identified using a phage-displayed random-peptide library. After three rounds of biopanning, ELISA showed that immunopositive phage clones specifically bound to mAb 70-21 but not to serum or purified IgG from naive mice. DNA sequencing of these phage clones showed a consensus sequence, QNNLGPR. Like mAb70-21, these phage-induced antisera also enhanced the DV infection of cells. In addition, indirect fluorescent assays showed phage-induced antisera bound to human rhabdomyosarcoma or Vero cells. Western blotting and immunoprecipitation analysis showed that phage-induced antisera recognized hsp 60 in BHK cell lysate. Moreover, the sera levels of antibodies against the synthetic peptide QNNLGPR correlated with the disease severity of dengue patients. Taken together, these results suggest that antibodies which recognized epitopes shared by pre-M of DV and hsp 60 of host cells may enhance DV infection and be involved in the development of DHF or DSS.

  2. Processing and functional display of the 86 kDa heterodimeric penicillin G acylase on the surface of phage fd

    NARCIS (Netherlands)

    Verhaert, R.M D; van Duin, J; Quax, Wim

    1999-01-01

    The large heterodimeric penicillin G acylase from Alcaligenes faecalis was displayed on the surface of phage fd. We fused the coding sequence (alpha subunit-internal peptide-beta subunit) to the gene of a phage coat protein. A modified g3p signal sequence was used to direct the polypeptide to the pe

  3. Phage-displayed peptides that mimic epitopes of hepatitis E virus capsid.

    Science.gov (United States)

    Larralde, Osmany; Petrik, Juraj

    2017-08-01

    Hepatitis E is an emerging zoonotic infection of increasing public health threat for the UK, especially for immunosuppressed individuals. A human recombinant vaccine has been licensed only in China and is not clear whether it protects against hepatitis E virus (HEV) genotype 3, the most prevalent in Europe. The aim of this study was to use phage display technology as a tool to identify peptides that mimic epitopes of HEV capsid (mimotopes). We identified putative linear and conformational mimotopes using sera from Scottish blood donors that have the immunological imprint of past HEV infection. Four mimotopes did not have homology with the primary sequence of HEV ORF2 capsid but competed effectively with a commercial HEV antigen for binding to anti-HEV reference serum. When the reactivity profile of each mimotope was compared with Wantai HEV-IgG ELISA, the most sensitive HEV immunoassay, mimotopes showed 95.2-100% sensitivity while the specificity ranged from 81.5 to 95.8%. PepSurf algorithm was used to map affinity-selected peptides onto the ORF2 crystal structure of HEV genotype 3, which predicted that these four mimototopes are clustered in the P domain of ORF2 capsid, near conformational epitopes of anti-HEV neutralising monoclonal antibodies. These HEV mimotopes may have potential applications in the design of structural vaccines and the development of new diagnostic tests.

  4. Three-dimensional display technologies.

    Science.gov (United States)

    Geng, Jason

    2013-01-01

    The physical world around us is three-dimensional (3D), yet traditional display devices can show only two-dimensional (2D) flat images that lack depth (i.e., the third dimension) information. This fundamental restriction greatly limits our ability to perceive and to understand the complexity of real-world objects. Nearly 50% of the capability of the human brain is devoted to processing visual information [Human Anatomy & Physiology (Pearson, 2012)]. Flat images and 2D displays do not harness the brain's power effectively. With rapid advances in the electronics, optics, laser, and photonics fields, true 3D display technologies are making their way into the marketplace. 3D movies, 3D TV, 3D mobile devices, and 3D games have increasingly demanded true 3D display with no eyeglasses (autostereoscopic). Therefore, it would be very beneficial to readers of this journal to have a systematic review of state-of-the-art 3D display technologies.

  5. Construction of Large Human Single-chain Antibody Phage Display Library

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    A large human naive single chain antibody (scFv) library is constructed from 60 healthy donors via phage display technique. During the period, some methods are employed to optimize the diversity, such as multi donors, different annealing temperature, half-nest PCR, and assembly by two-way fusion PCR. In this stud y, 78 electroporations resulted in 1010 library, diversity of which is assayed by enzyme fingerprint. The efficiency and diversity are all better than other rese arches.

  6. Identification of immunogenic proteins and generation of antibodies against Salmonella Typhimurium using phage display

    Directory of Open Access Journals (Sweden)

    Meyer Torsten

    2012-06-01

    Full Text Available Abstract Background Solely in Europoe, Salmonella Typhimurium causes more than 100,000 infections per year. Improved detection of livestock colonised with S. Typhimurium is necessary to prevent foodborne diseases. Currently, commercially available ELISA assays are based on a mixture of O-antigens (LPS or total cell lysate of Salmonella and are hampered by cross-reaction. The identification of novel immunogenic proteins would be useful to develop ELISA based diagnostic assays with a higher specificity. Results A phage display library of the entire Salmonella Typhimurium genome was constructed and 47 immunogenic oligopeptides were identified using a pool of convalescent sera from pigs infected with Salmonella Typhimurium. The corresponding complete genes of seven of the identified oligopeptids were cloned. Five of them were produced in E. coli. The immunogenic character of these antigens was validated with sera from pigs infeced with S. Tyhimurium and control sera from non-infected animals. Finally, human antibody fragments (scFv against these five antigens were selected using antibody phage display and characterised. Conclusion In this work, we identified novel immunogenic proteins of Salmonella Typhimurium and generated antibody fragments against these antigens completely based on phage display. Five immunogenic proteins were validated using a panel of positive and negative sera for prospective applications in diagnostics of Salmonela Typhimurium.

  7. Peptide Phage Display as a Tool for Drug Discovery: Targeting Membrane Receptors

    Directory of Open Access Journals (Sweden)

    Tomaz Bratkovic

    2011-01-01

    Full Text Available Ligands selected from phage-displayed random peptide libraries tend to be directed to biologically relevant sites on the surface of the target protein. Consequently, peptides derived from library screenings often modulate the target protein’s activity in vitro and in vivo and can be used as lead compounds in drug design and as alternatives to antibodies for target validation in both genomics and drug discovery. This review discusses the use of phage display to identify membrane receptor modulators with agonistic or antagonistic activities. Because isolating or producing recombinant membrane proteins for use as target molecules in library screening is often impossible, innovative selection strategies such as panning against whole cells or tissues, recombinant receptor ectodomains, or neutralizing antibodies to endogenous binding partners were devised. Prominent examples from a two-decade history of peptide phage display will be presented, focusing on the design of affinity selection experiments, methods for improving the initial hits, and applications of the identified peptides.

  8. Screening and Identification of a Novel Hepatocellular Carcinoma Cell Binding Peptide by Using a Phage Display Library

    Institute of Scientific and Technical Information of China (English)

    Xiaohua ZHU; Hua WU; Sha LUO; Zhiqun XIANYU; Dan ZHU

    2008-01-01

    The purpose of this study was to screen peptides that can specifically bind to human hepatocellular carcinoma (hHCC) cells using phage display of random peptide library in order to develope a peptide-based carrier for the diagnosis or therapy of hHCC. A peptide 12-mer phage display library was employed and 4 rounds of subtractive panning were performed using the hHCC cell line HepG2 as the target. After panning, the phages that specifically bound to and internalized in hHCC cells were selected. The selected phages demonstrated highly specific affinity to HepG2 cells analyzed by ELISA and immunofluorescence analysis. 57.3% of the selected phage clones displayed repeated sequence FLLEPHLMDTSM, and 4 amino acid residues, FLEP were extremely conservative. Based on the sequencing results, a 16-mer peptide (WH-16) was synthesized. The competitive ELISA showed that the binding of the phage clones displayed sequence FLLEPHLMDTSM to HepG2 cells was efficiently inhibited by WH-16. Our findings indicate that cellular binding of phage is mediated via its displayed peptide and the synthesized 16-mer peptide may have the potential to be a delivery Carrier in target diagnosis or therapy for hHCC.

  9. Application of phage display technique in molecular nuclear medicine%噬菌体展示技术在分子核医学中的应用

    Institute of Scientific and Technical Information of China (English)

    刘金剑; 刘鉴峰; 孟爱民

    2013-01-01

    噬菌体展示技术是研究分子间多种作用的技术,它能够在机理尚未明确的情况下研究蛋白质与蛋白质、蛋白质与多肽、蛋白质与核酸的相互作用,这种技术的一个关键优势是,它可以研究分子间的直接联系,得到不同亲和力的分子,一次性实现高效筛选多肽的目的.近年来随着噬菌体展示技术的成熟,该技术被广泛应用于生命科学研究的不同领域,如:单克隆抗体制备、多肽筛选、疫苗研制、基因治疗及细胞信号转导研究等.该文综述了噬菌体展示技术在分子核医学相关研究中的运用.%Phage display is a molecular diversity technology that allows the presentation of large peptide and protein libraries on the surface of filamentous phage.Phage display libraries permit the selection of peptides and proteins,including antibodies,with high affinity and specificity for almost any target.In recent years,along with phage display technology maturated,it has been widely used in screening tumor antibody library,the preparation of monoclonal antibody,polypeptide,drug design,gene therapy and cell signal transduction.This review serves as an introduction to phage display in molecular nuclear medicine,and recent application in display technology.

  10. Development of single chain variable fragment (scFv) antibodies against Xylella fastidiosa subsp. pauca by phage display.

    Science.gov (United States)

    Yuan, Qing; Jordan, Ramon; Brlansky, Ronald H; Istomina, Olga; Hartung, John

    2015-10-01

    Xylella fastidiosa is a member of the gamma proteobacteria. It is fastidious, insect-vectored and xylem-limited and causes a variety of diseases, some severe, on a wide range of economically important perennial crops, including grape and citrus. Antibody based detection assays are commercially available for X. fastidiosa, and are effective at the species, but not at the subspecies level. We have made a library of scFv antibody fragments directed against X. fastidiosa subsp. pauca strain 9a5c (citrus) by using phage display technology. Antibody gene repertoires were PCR-amplified using 23 primers for the heavy chain variable region (V(H)) and 21 primers for the light chain variable region (V(L)). The V(H) and V(L) were joined by overlap extension PCR, and then the genes of the scFv library were ligated into the phage vector pKM19. The library contained 1.2×10(7) independent clones with full-length scFv inserts. In each of 3cycles of affinity-selection with 9a5c, about 1.0×10(12) phage were used for panning with 4.1×10(6), 7.1×10(6), 2.1×10(7) phage recovered after the first, second and third cycles, respectively. Sixty-six percent of clones from the final library bound X. fastidiosa 9a5c in an ELISA. Some of these scFv antibodies recognized strain 9a5c and did not recognize X. fastidiosa strains that cause Pierce's disease of grapevine.

  11. Affinity peptide developed by phage display selection for targeting gastric cancer

    Institute of Scientific and Technical Information of China (English)

    Wen-Jie Zhang; Yan-Xia Sui; Arun Budha; Jian-Bao Zheng; Xue-Jun Sun; Ying-Chun Hou; Thomas D Wang; Shao-Ying Lu

    2012-01-01

    AIM:To develop an affinity peptide that binds to gastric cancer used for the detection of early gastric cancer.METHODS:A peptide screen was performed by biopanning the PhD-12 phage display library,clearing non-specific binders against tumor-adjacent normal appearing gastric mucosa and obtaining selective binding against freshly harvested gastric cancer tissues.Tumortargeted binding of selected peptides was confirmed by bound phage counts,enzyme-linked immunosorbent assay,competitive inhibition,fluorescence microscopy and semi-quantitative analysis on immunohistochemistry using different types of cancer tissues.RESULTS:Approximately 92.8% of the non-specific phage clones were subtracted from the original phage library after two rounds of biopanning against normalappearing gastric mucosa.After the third round of positive screening,the peptide sequence AADNAKTKSFPV (AAD) appeared in 25% (12/48) of the analyzed phages.For the control peptide,these values were 6.8 ± 2.3,5.1 ± 1.7,3.5 ± 2.1,4.6 ± 1.9 and 1.1 ± 0.5,respectively.The values for AAD peptide were statistically significant (P < 0.01) for gastric cancer as compared with other histological classifications and control peptide.CONCLUSION:A novel peptide is discovered to have a specific binding activity to gastric cancer,and can be used to distinguish neoplastic from normal gastric mucosa,demonstrating the potential for early cancer detection on endoscopy.

  12. Phage display of the serpin alpha-1 proteinase inhibitor randomized at consecutive residues in the reactive centre loop and biopanned with or without thrombin.

    Directory of Open Access Journals (Sweden)

    Benjamin M Scott

    Full Text Available In spite of the power of phage display technology to identify variant proteins with novel properties in large libraries, it has only been previously applied to one member of the serpin superfamily. Here we describe phage display of human alpha-1 proteinase inhibitor (API in a T7 bacteriophage system. API M358R fused to the C-terminus of T7 capsid protein 10B was directly shown to form denaturation-resistant complexes with thrombin by electrophoresis and immunoblotting following exposure of intact phages to thrombin. We therefore developed a biopanning protocol in which thrombin-reactive phages were selected using biotinylated anti-thrombin antibodies and streptavidin-coated magnetic beads. A library consisting of displayed API randomized at residues 357 and 358 (P2-P1 yielded predominantly Pro-Arg at these positions after five rounds of thrombin selection; in contrast the same degree of mock selection yielded only non-functional variants. A more diverse library of API M358R randomized at residues 352-356 (P7-P3 was also probed, yielding numerous variants fitting a loose consensus of DLTVS as judged by sequencing of the inserts of plaque-purified phages. The thrombin-selected sequences were transferred en masse into bacterial expression plasmids, and lysates from individual colonies were screening for API-thrombin complexing. The most active candidates from this sixth round of screening contained DITMA and AAFVS at P7-P3 and inhibited thrombin 2.1-fold more rapidly than API M358R with no change in reaction stoichiometry. Deep sequencing using the Ion Torrent platform confirmed that over 800 sequences were significantly enriched in the thrombin-panned versus naïve phage display library, including some detected using the combined phage display/bacterial lysate screening approach. Our results show that API joins Plasminogen Activator Inhibitor-1 (PAI-1 as a serpin amenable to phage display and suggest the utility of this approach for the selection

  13. Construction and diversity analysis of a murine IgE phage surface display library

    Institute of Scientific and Technical Information of China (English)

    LIZONGDONG; MINGYEH

    1997-01-01

    To make further investigation of the IgE antibody repertoire in Trichosanthin (TCS) allergic responses,a murine IgE phage surface display library was constructed (3.0×105 independent clones).We first constructed the Vε cDNA library (4.6×105 independent clones) and Vκ cDNA library (3.0×105 independent clones).Then,the Vε and Vκgene segments were amplified from both libraries by PCR respectively,and assembled into Fab fragment by SOE PCR.The phage library containing Fabs was thus constructed.The diversity of Vε from this library was analyzed and proved.Fab clones with high specificity to TCS have been screened out.

  14. Blocking peptides against HBV: PreS1 protein selected from a phage display library

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Wei; Liu, Yang; Zu, Xiangyang; Jin, Rui [State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071 (China); Xiao, Gengfu, E-mail: xiaogf@wh.iov.cn [State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071 (China)

    2011-09-09

    Highlights: {yields} Successfully selected specific PreS1-interacting peptides by using phage displayed library. {yields} Alignment of the positive phage clones revealed a consensus PreS1 binding motif. {yields} A highly enriched peptide named P7 had a strong binding ability for PreS1. {yields} P7 could block PreS1 attachment. -- Abstract: The PreS1 protein is present on the outermost part of the hepatitis B virus (HBV) surface and has been shown to have a pivotal function in viral infectivity and assembly. The development of reagents with high affinity and specificity for PreS1 is of great significance for early diagnosis and treatment of HBV infection. A phage display library of dodecapeptide was screened for interactions with purified PreS1 protein. Alignment of the positive phage clones revealed a putative consensus PreS1 binding motif of HX{sub n}HX{sub m}HP/R. Moreover, a peptide named P7 (KHMHWHPPALNT) was highly enriched and occurred with a surprisingly high frequency of 72%. A thermodynamic study revealed that P7 has a higher binding affinity to PreS1 than the other peptides. Furthermore, P7 was able to abrogate the binding of HBV virions to the PreS1 antibody, suggesting that P7 covers key functional sites on the native PreS1 protein. This newly isolated peptide may, therefore, be a new therapeutic candidate for the treatment of HBV. The consensus motif could be modified to deliver imaging, diagnostic, and therapeutic agents to tissues affected by HBV.

  15. 噬菌体抗体库筛选技术%Screening Isolates from Antibody Phage-display Libraries

    Institute of Scientific and Technical Information of China (English)

    高鹏; 胡立勇; 钱钰; 徐晨

    2012-01-01

    For almost 20 years, phage display antibody (Ab) libraries screening have been widely used in antibody screen, clinical diagnostic, treatment of disease and basic research areas. Rapid identification of the autofit monoclonal antibody, and reliable and efficient data management and analysis are very important in the application of this technology. This review described reported methods for high-throughput screening of antibody phage-display libraries and further more gave a brief introduction to data management.%在过去的20年中,噬菌体抗体库筛选技术被广泛的应用于抗体筛选、疾病治疗,临床诊断以及基础研究之中.在该技术的应用过程中,快速有效的筛选出最适合的单克隆抗体并进行可靠和高效的数据管理和分析是十分重要的.文中总结了近年来噬菌体抗体库的高通量筛选方法并且对数据管理做了简要介绍.

  16. Phage Display Screening for Tumor Necrosis Factor-α-Binding Peptides: Detection of Inflammation in a Mouse Model of Hepatitis

    Directory of Open Access Journals (Sweden)

    Coralie Sclavons

    2013-01-01

    Full Text Available TNF-α is one of the most abundant cytokines produced in many inflammatory and autoimmune conditions such as multiple sclerosis, chronic hepatitis C, or neurodegenerative diseases. These pathologies remain difficult to diagnose and consequently difficult to treat. The aim of this work is to offer a new diagnostic tool by seeking new molecular probes for medical imaging. The target-specific part of the probe consists here of heptameric peptides selected by the phage display technology for their affinity for TNF-α. Several affinity tests allowed isolating 2 peptides that showed the best binding capacity to TNF-α. Finally, the best peptide was synthesized in both linear and cyclic forms and tested on the histological sections of concanavalin-A-(ConA-treated mice liver. In this well-known hepatitis mouse model, the best results were obtained with the cyclic form of peptide 2, which allowed for the staining of inflamed areas in the liver. The cyclic form of peptide 2 (2C was, thus, covalently linked to iron oxide nanoparticles (magnetic resonance imaging (MRI contrast agent and tested in the ConA-induced hepatitis mouse model. The vectorized nanoparticles allowed for the detection of inflammation as well as of the free peptide. These ex vivo results suggest that phage display-selected peptides can direct imaging contrast agents to inflammatory areas.

  17. Rapid Selection of Phage Se-scFv with GPX Activity via Combination of Phage Display Antibody Library with Chemical Modification

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Glutathione peroxidase (GPX) plays an important role in scavenging reactive oxygen species. A series of catalytic antibodies with GPX activity have been generated by the authors of this study. To obtain humanized catalytic antibodies, the phage-displayed human antibody library was used to select novel antibodies by repetitive screening. Phage antibodies, scFv-B8 and scFv-H6 with the GSH-binding site, were obtained from the library by enzyme-linked immunosorbent assay(ELISA) analysis with 4 rounds of selection against their respective haptens, S-2,4-dinitriphenyl t-butyl ester(GSH-s-DNP-Bu) and S-2,4-dinitriphenyl t-hexyl ester(GSH-s-DNP-He). Nevertheless, several studies need to be conducted to determine whether scFv-B8 and scFv-H6 possess GPX activity. To enhance the speed of the selection, selenocysteine(Sec, the catalytic group of GPX) was incorporated directly into the phages, scFv-B8 and scFv-H6, by chemical mutation to form the phages Se-scFv-B8 and Se-scFv-H6. The GPX activities were found to be 3012 units/μmol and 2102 units/μmol, respectively. To improve the GPX activity of the phage Se-scFv-B8, DNA shuffling was used to construct a secondary library and another positive phage antibody scFv-B9 was screened out by another panning against GSH-s-DNP-Bu. When Sec was incorporated via chemical mutation into the phage antibody scFv-B9, its GPX activity reached 3560 units/μmol, which is 1.17-fold higher than the phage antibody Se-scFv-B8and almost approached the order of magnitude of native GPX. The rapid selection is the prerequisite for generating humanized Se-scFv with GPX activity.

  18. Discovery and Characterization of Phage Display-Derived Human Monoclonal Antibodies against RSV F Glycoprotein.

    Directory of Open Access Journals (Sweden)

    Zhifeng Chen

    Full Text Available Respiratory syncytial virus (RSV is a leading cause of lower respiratory tract infection in infants, the elderly and in immunosuppressed populations. The vast majority of neutralizing antibodies isolated from human subjects target the RSV fusion (F glycoprotein, making it an attractive target for the development of vaccines and therapeutic antibodies. Currently, Synagis® (palivizumab is the only FDA approved antibody drug for the prevention of RSV infection, and there is a great need for more effective vaccines and therapeutics. Phage display is a powerful tool in antibody discovery with the advantage that it does not require samples from immunized subjects. In this study, Morphosys HuCAL GOLD® phage libraries were used for panning against RSV prefusion and postfusion F proteins. Panels of human monoclonal antibodies (mAbs against RSV F protein were discovered following phage library panning and characterized. Antibodies binding specifically to prefusion or postfusion F proteins and those binding both conformations were identified. 3B1 is a prototypic postfusion F specific antibody while 2E1 is a prototypic prefusion F specific antibody. 2E1 is a potent broadly neutralizing antibody against both RSV A and B strains. Epitope mapping experiments identified a conformational epitope spanning across three discontinuous sections of the RSV F protein, as well as critical residues for antibody interaction.

  19. Phage-displayed peptides selected for binding to Campylobacter jejuni are antimicrobial.

    Science.gov (United States)

    Bishop-Hurley, Sharon L; Rea, Philippa J; McSweeney, Christopher S

    2010-10-01

    In developed countries, Campylobacter jejuni is a leading cause of zoonotic bacterial gastroenteritis in humans with chicken meat implicated as a source of infection. Campylobacter jejuni colonises the lower gastrointestinal tract of poultry and during processing is spread from the gastrointestinal tract onto the surface of dressed carcasses. Controlling or eliminating C.jejuni on-farm is considered to be one of the best strategies for reducing human infection. Molecules on the cell surface of C.jejuni interact with the host to facilitate its colonisation and persistence in the gastrointestinal tract of poultry. We used a subtractive phage-display protocol to affinity select for peptides binding to the cell surface of a poultry isolate of C.jejuni with the aim of finding peptides that could be used to control this microorganism in chickens. In total, 27 phage peptides, representing 11 unique clones, were found to inhibit the growth of C.jejuni by up to 99.9% in vitro. One clone was bactericidal, reducing the viability of C.jejuni by 87% in vitro. The phage peptides were highly specific. They completely inhibited the growth of two of the four poultry isolates of C.jejuni tested with no activity detected towards other Gram-negative and Gram-positive bacteria.

  20. A novel peptide, selected from phage display library of random peptides, can efficiently target into human breast cancer cell

    Institute of Scientific and Technical Information of China (English)

    DONG Jian; LIU WeiQing; JIANG AiMei; ZHANG KeJian; CHEN MingQing

    2008-01-01

    To develop a targeting vector for breast cancer biotherapy, MDA-MB-231 cell, a human breast cancer cell line, was co-cultured with pC89 (9 aa) phage display library of random peptides. In multiple inde-pendent peptide-presenting phage screening trials, subtilisin was used as a protease to inactivate ex-tra-cellular phages. The internalized phages were collected by cell lysising and amplified in E. coli XLI-Blue. Through five rounds of selection, the peptide-presenting phages which could be internalized in MDA-MB-231 cells were isolated. A comparison was made between internalization capacities of pep-tide-presenting phages isolated from MDA-MB-231 cells and RGD-integrin binding phage by cocultur-ing them with other human tumor cell lines and normal cells. The nucleotide sequences of isolated peptide-presenting phages were then determined by DNA sequencing. To uncover whether phage coat protein or amino acid order was required for the character of the peptide to MDA-MB-231 cells, three peptides were synthesized. They are CASPSGALRSC, ASPSGALRS and CGVIFDHSVPC (the shifted sequence of CASPSGALRSC), and after coculturing them with different cell lines, their targeting ca-pacities to MDA-MB-231 cells were detected. These data suggested that the internalization process was highly selective, and capable of capturing a specific peptide from parent peptide variants. Moreover, the targeting internalization event of peptides was an amino acid sequence dependent manner. The results demonstrated the feasibility of using phage display library of random peptides to develop new targeting system for intracellular delivery of macromolecules, and the peptide we obtained might be modified as a targeting vector for breast cancer gene therapy.

  1. Phage display:development of nanocarriers for targeted drug delivery to the brain

    Institute of Scientific and Technical Information of China (English)

    Babak Bakhshinejad; Marzieh Karimi; Mohammad Khalaj-Kondori

    2015-01-01

    The blood brain barrier represents a formidable obstacle for the transport of most systemati-cally administered neurodiagnostics and neurotherapeutics to the brain. Phage display is a high throughput screening strategy that can be used for the construction of nanomaterial peptide libraries. These libraries can be screened for ifnding brain targeting peptide ligands. Surface func-tionalization of a variety of nanocarriers with these brain homing peptides is a sophisticated way to develop nanobiotechnology-based drug delivery platforms that are able to cross the blood brain barrier. These efifcient drug delivery systems raise our hopes for the diagnosis and treatment of various brain disorders in the future.

  2. Identification of Novel Macropinocytosing Human Antibodies by Phage Display and High-Content Analysis.

    Science.gov (United States)

    Ha, K D; Bidlingmaier, S M; Su, Y; Lee, N-K; Liu, B

    2017-01-01

    Internalizing antibodies have great potential for the development of targeted therapeutics. Antibodies that internalize via the macropinocytosis pathway are particularly promising since macropinocytosis is capable of mediating rapid, bulk uptake and is selectively upregulated in many cancers. We hereby describe a method for identifying antibodies that internalize via macropinocytosis by screening phage-displayed single-chain antibody selection outputs with an automated fluorescent microscopy-based high-content analysis platform. Furthermore, this method can be similarly applied to other endocytic pathways if other fluorescent, pathway-specific, soluble markers are available. © 2017 Elsevier Inc. All rights reserved.

  3. Thermal Stability of RNA Phage Virus-Like Particles Displaying Foreign Peptides

    Directory of Open Access Journals (Sweden)

    Peabody David S

    2011-05-01

    Full Text Available Abstract Background To be useful for genetic display of foreign peptides a viral coat protein must tolerate peptide insertions without major disruption of subunit folding and capsid assembly. The folding of the coat protein of RNA phage MS2 does not normally tolerate insertions in its AB-loop, but an engineered single-chain dimer readily accepts them as long as they are restricted to one of its two halves. Results Here we characterize the effects of peptide insertions on the thermal stabilities of MS2 virus-like particles (VLPs displaying a variety of different peptides in one AB-loop of the coat protein single-chain dimer. These particles typically denature at temperatures around 5-10°C lower than unmodified VLPs. Even so, they are generally stable up to about 50°C. VLPs of the related RNA phage PP7 are cross-linked with intersubunit disulfide bonds and are therefore significantly more stable. An AB-loop insertion also reduces the stability of PP7 VLPs, but they only begin to denature above about 70°C. Conclusions VLPs assembled from MS2 single-chain dimer coat proteins with peptide insertions in one of their AB-loops are somewhat less stable than the wild-type particle, but still resist heating up to about 50°C. Because they possess disulfide cross-links, PP7-derived VLPs provide an alternate platform with even higher stability.

  4. Unique secreted–surface protein complex of Lactobacillus rhamnosus, identified by phage display

    Science.gov (United States)

    Gagic, Dragana; Wen, Wesley; Collett, Michael A; Rakonjac, Jasna

    2013-01-01

    Proteins are the most diverse structures on bacterial surfaces; hence, they are candidates for species- and strain-specific interactions of bacteria with the host, environment, and other microorganisms. Genomics has decoded thousands of bacterial surface and secreted proteins, yet the function of most cannot be predicted because of the enormous variability and a lack of experimental data that would allow deduction of function through homology. Here, we used phage display to identify a pair of interacting extracellular proteins in the probiotic bacterium Lactobacillus rhamnosus HN001. A secreted protein, SpcA, containing two bacterial immunoglobulin-like domains type 3 (Big-3) and a domain distantly related to plant pathogen response domain 1 (PR-1-like) was identified by screening of an L. rhamnosus HN001 library using HN001 cells as bait. The SpcA-“docking” protein, SpcB, was in turn detected by another phage display library screening, using purified SpcA as bait. SpcB is a 3275-residue cell-surface protein that contains general features of large glycosylated Serine-rich adhesins/fibrils from gram-positive bacteria, including the hallmark signal sequence motif KxYKxGKxW. Both proteins are encoded by genes within a L. rhamnosus-unique gene cluster that distinguishes this species from other lactobacilli. To our knowledge, this is the first example of a secreted-docking protein pair identified in lactobacilli. PMID:23233310

  5. Generation of human antibody fragments against Streptococcus mutans using a phage display chain shuffling approach

    Directory of Open Access Journals (Sweden)

    Barth Stefan

    2005-01-01

    Full Text Available Abstract Background Common oral diseases and dental caries can be prevented effectively by passive immunization. In humans, passive immunotherapy may require the use of humanized or human antibodies to prevent adverse immune responses against murine epitopes. Therefore we generated human single chain and diabody antibody derivatives based on the binding characteristics of the murine monoclonal antibody Guy's 13. The murine form of this antibody has been used successfully to prevent Streptococcus mutans colonization and the development of dental caries in non-human primates, and to prevent bacterial colonization in human clinical trials. Results The antibody derivatives were generated using a chain-shuffling approach based on human antibody variable gene phage-display libraries. Like the parent antibody, these derivatives bound specifically to SAI/II, the surface adhesin of the oral pathogen S. mutans. Conclusions Humanization of murine antibodies can be easily achieved using phage display libraries. The human antibody fragments bind the antigen as well as the causative agent of dental caries. In addition the human diabody derivative is capable of aggregating S. mutans in vitro, making it a useful candidate passive immunotherapeutic agent for oral diseases.

  6. Analysis of the Resveratrol-binding Protein using Phage-displayed Random Peptide Library

    Institute of Scientific and Technical Information of China (English)

    Lei FENG; Jian JIN; Lian-Feng ZHANG; Ting YAN; Wen-Yi TAO

    2006-01-01

    Resveratrol, a plant polyphenol, is found in significant amounts in the skin of grapes and in some traditional herbs. It is reported to exert different biological activities, such as inhibiting lipid peroxidation,scavenging free radicals, inhibiting platelet aggregation, and anticancer activity. In order to screen the resveratrol-binding proteins, we synthesized biotinylated resveratrol, purified by liquid chromatography and immobilized it into streptavidin-coated microplate wells. 3-(4,5-Demethylthiazol-)-2,5-diphenyl tetrazolium bromide assay showed little change in the anticancer activity of biotinylated resveratrol in vitro. A random library of phage-displayed peptides was screened for binding to immobilized resveratrol to isolate resveratrolbinding proteins. Several peptides were found to bind to resveratrol specifically, which was proven by enzyme-linked immunosorbent assay. Through amino acid sequence analysis of the selected peptides and human proteins using the BLAST program, the results showed that resveratrol has an affinity for various proteins such as breast cancer-associated antigen, breast cancer resistance protein, death-associated transcription factor, and human cyclin-dependent kinase. These results demonstrate that our study provides a feasible method for the study of binding proteins of natural compounds using a phage-displayed random peptide library.

  7. Antitumor activity of endogenous mFlt4 displayed on a T4 phage nanoparticle surface

    Institute of Scientific and Technical Information of China (English)

    Shun-xiang REN; Zhao-jun REN; Min-yi ZHAO; Xiao-bin WANG; Shu-guang ZUO; Feng YU

    2009-01-01

    Aim: Flt4 plays a key role in promoting tumor metastasis by stimulating solid tumor lymphangiogenesis. In this study,mouse Flt4 (mFlt4) was displayed on T4 phage in order to explore the feasibility of breaking immune tolerance to "selfantigens" and to evaluate the phage's antitumor activity.Methods: A T4 phage nanometer particle expressing mFlt4 on the surface was constructed for evaluation as a recombinant vaccine. The presence of the mFlt4 gene in the T4-mFlt4 recombinant vaccine was verified by PCR and Western blot analysis. The immunotherapeutic potential of T4-mFlt4 was tested in mice injected with Lewis lung carcinoma (LLC) cells. AntiFlt4 antibody producing B cells were detected by ELISPOT. The effects of T4-mFlt4 on lymphatic metastasis and lymphangiogenesis were investigated in a mouse antimetastasis assay and by Flt4 and CD105 immunohistochemistry.Results: The T4-mFlt4 recombinant vaccine demonstrated antitumor activity and elicited autoantibodies against mFlt4.Mice carrying LLC-derived tumors exhibited prolonged survival when given the vaccine compared with control-treated animals. The vaccine also inhibited lymphangiogenesis and tumor metastasis in the mouse models. However, T4-mFlt4 was not observed to inhibit tumor growth.Conclusion: The T4-mFlt4 recombinant vaccine induced protective antitumor immunity and antimetastasis against LLC.Induction of an autoimmune response directed against tumor progression merits further study as a new strategy for immunotherapy in cancer.

  8. Phage display against corneal epithelial cells produced bioactive peptides that inhibit Aspergillus adhesion to the corneas.

    Directory of Open Access Journals (Sweden)

    Ge Zhao

    Full Text Available Dissection of host-pathogen interactions is important for both understanding the pathogenesis of infectious diseases and developing therapeutics for the infectious diseases like various infectious keratitis. To enhance the knowledge about pathogenesis infectious keratitis, a random 12-mer peptide phage display library was screened against cultured human corneal epithelial cells (HCEC. Fourteen sequences were obtained and BLASTp analysis showed that most of their homologue counterparts in GenBank were for defined or putative proteins in various pathogens. Based on known or predicted functions of the homologue proteins, ten synthetic peptides (Pc-A to Pc-J were measured for their affinity to bind cells and their potential efficacy to interfere with pathogen adhesion to the cells. Besides binding to HCEC, most of them also bound to human corneal stromal cells and umbilical endothelial cells to different extents. When added to HCEC culture, the peptides induced expression of MyD88 and IL-17 in HCEC, and the stimulated cell culture medium showed fungicidal potency to various extents. While peptides Pc-C and Pc-E inhibited Aspergillus fumigatus (A.f adhesion to HCEC in a dose-dependent manner, the similar inhibition ability of peptides Pc-A and Pc-B required presence of their homologue ligand Alb1p on A.f. When utilized in an eyeball organ culture model and an in vivo A.f keratitis model established in mouse, Pc-C and Pc-E inhibited fungal adhesion to corneas, hence decreased corneal disruption caused by inflammatory infiltration. Affinity pull-down of HCEC membrane proteins with peptide Pc-C revealed several molecules as potential receptors for this peptide. In conclusion, besides proving that phage display-selected peptides could be utilized to interfere with adhesion of pathogens to host cells, hence could be exploited for managing infectious diseases including infectious keratitis, we also proposed that the phage display technique and the

  9. High affinity antibodies against Lex and sialyl Lex from a phage display library.

    Science.gov (United States)

    Dinh, Q; Weng, N P; Kiso, M; Ishida, H; Hasegawa, A; Marcus, D M

    1996-07-15

    Our previous studies of seven murine mAbs against the carbohydrate Lex Ag demonstrated that they were all encoded by VH441 and V kappa 24B. To obtain higher affinity Abs, and to ascertain whether their L chains could be encoded by other genes, we constructed a phage display library in a modified pComb 8 vector. The library contained random L chains, and Fd segments enriched in VH domains encoded by the VHX24 gene family. We selected phage with an Lex-BSA Ag, and obtained two Fab mAbs, clones 23 and 24, whose affinities were more than 100-fold higher than hybridoma mAb PM81. Both new mAbs were encoded by VH441, and their L chains were encoded by genes of the V kappa Ox1 and V kappa 9 families. In contrast to hybridoma mAb PM81, which binds only Lex, clones 23 and 24 bound sialyl Lex (SLex) as well as Lex, and clone 23 also binds the backbone carbohydrate structure nLacCer. Analysis of the binding of these three mAbs to synthetic glycolipids that contained structural modifications indicated that they recognize different aspects of the Lex structure, and suggested that they bind to limited regions of the oligosaccharide.

  10. Localization of melanin in conidia of Alternaria alternata using phage display antibodies.

    Science.gov (United States)

    Carzaniga, Raffaella; Fiocco, Daniela; Bowyer, Paul; O'Connell, Richard J

    2002-03-01

    Melanins derived from 1,8-dihydroxynaphthalene (DHN) are important for the pathogenicity and survival of fungi causing disease in both plants and animals. However, precise information on their location within fungal cell walls is lacking. To obtain antibodies for the immunocytochemical localization of melanin, 83 phage antibodies binding to 1,8-DHN were selected from a naive semisynthetic single-chain Fv (scFv) phage display library. Sequence analysis of the heavy chain binding domains of 17 antibodies showed a high frequency of positively charged amino acids. One antibody, designated M1, was characterized in detail. M1 bound specifically to 1,8-DHN in competitive inhibition enzyme-linked immunosorbent assays, showing no cross-reaction with nine structurally related phenolic compounds. Epitope recognition required two hydroxyl groups in a 1,8 configuration. M1 also bound to naturally occurring melanin isolated from mycelia of Alternaria alternata, suggesting that epitopes remain accessible in polymerized melanin. Transmission electron microscopy-immunogold labeling, using M1 in the form of soluble scFv fragments, showed that melanin was located in the septa and outer (primary) walls of wild-type A. alternata conidia, but not those of an albino mutant, AKT88-1. The M1 antibody provides a new tool for detecting melanized pathogens in plant and animal tissues and for precisely mapping the distribution of the polymer within spores, appressoria, and hyphae.

  11. Novel ZnO-binding peptides obtained by the screening of a phage display peptide library

    Energy Technology Data Exchange (ETDEWEB)

    Golec, Piotr [Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Laboratory of Molecular Biology (affiliated with the University of Gdansk) (Poland); Karczewska-Golec, Joanna [University of Gdansk and Medical University of Gdansk, Laboratory of Molecular Bacteriology, Intercollegiate Faculty of Biotechnology (Poland); Los, Marcin; Wegrzyn, Grzegorz, E-mail: wegrzyn@biotech.univ.gda.pl [University of Gdansk, Department of Molecular Biology (Poland)

    2012-11-15

    Zinc oxide (ZnO) is a semiconductor compound with a potential for wide use in various applications, including biomaterials and biosensors, particularly as nanoparticles (the size range of ZnO nanoparticles is from 2 to 100 nm, with an average of about 35 nm). Here, we report isolation of novel ZnO-binding peptides, by screening of a phage display library. Interestingly, amino acid sequences of the ZnO-binding peptides reported in this paper and those described previously are significantly different. This suggests that there is a high variability in sequences of peptides which can bind particular inorganic molecules, indicating that different approaches may lead to discovery of different peptides of generally the same activity (e.g., binding of ZnO) but having various detailed properties, perhaps crucial under specific conditions of different applications.

  12. Identification and in vitro characterization of phage-displayed VHHs targeting VEGF

    DEFF Research Database (Denmark)

    Farajpour, Zahra; Rahbarizadeh, Fatemeh; Kazemi, Bahram;

    2014-01-01

    Vascular endothelial growth factor (VEGF) is a potential target for cancer treatment because of its role in angiogenesis and its overexpression in most human cancers. Currently, anti-VEGF antibodies have been shown to be promising tools for therapeutic applications. However, large size, poor tumor......-targeting purposes. The present study was undertaken to generate and characterize anti-VEGF VHHs from an immune VHH library using phage display. Four rounds of panning were performed, and selected VHHs were characterized using various immunological techniques. Assessment of the antigenic profile of VHHs was done......, significantly inhibited the endothelial cell growth in a dose-dependent manner. Taken together, our results indicate that ZFR-5 and other VHHs may be promising tools in cancer research and treatment....

  13. Identification of binding peptides of the ADAM15 disintegrin domain using phage display

    Indian Academy of Sciences (India)

    Jing Wu; Min-Chen Wu; Lian-Fen Zhang; Jian-Yong Lei; Lei Feng; Jian Jin

    2009-06-01

    ADAM15 plays an important role in tumour development by interacting with integrins. In this study, we investigated the target peptides of the ADAM15 disintegrin domain. First, we successfully produced the recombinant human ADAM15 disintegrin domain (RADD) that could inhibit melanoma cell adhesion by using Escherichia coli. Second, four specific binding peptides (peptides A, B, C, and D) were selected using a phage display 12-mer peptide library. The screening protocol involved 4 rounds of positive panning on RADD and 2 rounds of subtractive selection with streptavidin. By using the BLAST software and a relevant protein database, integrin v3 was found to be homologous to peptide A. Synthetic peptide A had a highly inhibitory effect on RADD–integrin v3 binding. The results demonstrate the potential application of short peptides for disrupting high-affinity ADAM–integrin interactions.

  14. Isolation of Osteosarcoma-Associated Human Antibodies from a Combinatorial Fab Phage Display Library

    Directory of Open Access Journals (Sweden)

    Carmela Dantas-Barbosa

    2009-01-01

    Full Text Available Osteosarcoma, a highly malignant disease, is the most common primary bone tumor and is frequently found in children and adolescents. In order to isolate antibodies against osteosarcoma antigens, a combinatorial osteosarcoma Fab library displayed on the surface of phages was used. After three rounds of selection on the surface of tumor cells, several osteosarcoma-reactive Fabs were detected. From these Fabs, five were better characterized, and despite having differences in their VH (heavy chain variable domain and Vκ (kappa chain variable domain regions, they all bound to a protein with the same molecular mass. Further analysis by cell ELISA and immunocytochemistry suggested that the Fabs recognize a membrane-associated tumor antigen expressed in higher amounts in neoplasic cells than in normal tissue. These results suggest that the human Fabs selected in this work are a valuable tool for the study of this neoplasia.

  15. Phage-displayed peptide library screening for preferred human substrate peptide sequences for transglutaminase 7.

    Science.gov (United States)

    Kuramoto, Katsuma; Yamasaki, Risa; Shimizu, Yoshitaka; Tatsukawa, Hideki; Hitomi, Kiyotaka

    2013-09-01

    Transglutaminases are a family of enzymes that catalyze cross-linking reactions between proteins. Among the members, there is currently no information regarding the substrate preferences of transglutaminase 7 (TG7), that would clarify its physiological significance. We previously obtained several highly reactive substrate peptide sequences of transglutaminases from a random peptide library. In this study, we screened for preferred substrate sequences for TG7 from a phage-displayed 12-mer peptide library. The most preferred sequence was selected based on reactivity and isozyme specificity. We firstly exhibited the tendency for the preference of substrate sequence for TG7. Then, using the most efficient peptide, Z3S, we established an in vitro assay system to assess enzymatic activity of TG7.

  16. Identification of Novel Immunogenic Proteins of Neisseria gonorrhoeae by Phage Display.

    Directory of Open Access Journals (Sweden)

    Daniel O Connor

    Full Text Available Neisseria gonorrhoeae is one of the most prevalent sexually transmitted diseases worldwide with more than 100 million new infections per year. A lack of intense research over the last decades and increasing resistances to the recommended antibiotics call for a better understanding of gonococcal infection, fast diagnostics and therapeutic measures against N. gonorrhoeae. Therefore, the aim of this work was to identify novel immunogenic proteins as a first step to advance those unresolved problems. For the identification of immunogenic proteins, pHORF oligopeptide phage display libraries of the entire N. gonorrhoeae genome were constructed. Several immunogenic oligopeptides were identified using polyclonal rabbit antibodies against N. gonorrhoeae. Corresponding full-length proteins of the identified oligopeptides were expressed and their immunogenic character was verified by ELISA. The immunogenic character of six proteins was identified for the first time. Additional 13 proteins were verified as immunogenic proteins in N. gonorrhoeae.

  17. Identification of Novel Immunogenic Proteins of Neisseria gonorrhoeae by Phage Display.

    Science.gov (United States)

    Connor, Daniel O; Zantow, Jonas; Hust, Michael; Bier, Frank F; von Nickisch-Rosenegk, Markus

    2016-01-01

    Neisseria gonorrhoeae is one of the most prevalent sexually transmitted diseases worldwide with more than 100 million new infections per year. A lack of intense research over the last decades and increasing resistances to the recommended antibiotics call for a better understanding of gonococcal infection, fast diagnostics and therapeutic measures against N. gonorrhoeae. Therefore, the aim of this work was to identify novel immunogenic proteins as a first step to advance those unresolved problems. For the identification of immunogenic proteins, pHORF oligopeptide phage display libraries of the entire N. gonorrhoeae genome were constructed. Several immunogenic oligopeptides were identified using polyclonal rabbit antibodies against N. gonorrhoeae. Corresponding full-length proteins of the identified oligopeptides were expressed and their immunogenic character was verified by ELISA. The immunogenic character of six proteins was identified for the first time. Additional 13 proteins were verified as immunogenic proteins in N. gonorrhoeae.

  18. Design of phage-displayed cystine-stabilized mini-protein libraries for proteinaceous binder engineering.

    Science.gov (United States)

    Chang, Hung-Ju; Yang, An-Suei

    2014-01-01

    Cystine-stabilized mini-proteins are important scaffolds in the combinatorial search of binders for molecular recognition. The structural determinants of a cystine-stabilized scaffold are the critical residues determining the formation of the native disulfide-bonding configuration, and thus should remain unchanged in the combinatorial libraries so as to allow a large portion of the library sequences to be compatible with the scaffold structure. A high-throughput molecular evolution procedure has been developed to select and screen for the polypeptide sequences folding into a specific cystine-stabilized structure. Patterns of sequence preference that emerge from the resultant sequence profiles provide structural determinant information, which facilitates the designs of combinatorial libraries for combinatorial approaches as in phage display. This methodology enables artificial cystine-stabilized proteins to be engineered with enhanced folding and binding properties.

  19. Selection of Ceratitis capitata (Diptera: Tephritidae) specific recombinant monoclonal phage display antibodies for prey detection analysis.

    Science.gov (United States)

    Monzó, César; Urbaneja, Alberto; Ximénez-Embún, Miguel; García-Fernández, Julia; García, José Luis; Castañera, Pedro

    2012-01-01

    Several recombinant antibodies against the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), one of the most important pests in agriculture worldwide, were selected for the first time from a commercial phage display library of human scFv antibodies. The specificity and sensitivity of the selected recombinant antibodies were compared with that of a rabbit polyclonal serum raised in parallel using a wide range of arthropod species as controls. The selected recombinant monoclonal antibodies had a similar or greater specificity when compared with classical monoclonal antibodies. The selected recombinant antibodies were successfully used to detect the target antigen in the gut of predators and the scFv antibodies were sequenced and compared. These results demonstrate the potential for recombinant scFv antibodies to be used as an alternative to the classical monoclonal antibodies or even molecular probes in the post-mortem analysis studies of generalist predators.

  20. Modular and aggregation resistant Vh antibodies from a phage display library

    DEFF Research Database (Denmark)

    Friis, Niels Anton; Mandrup, Ole Aalund; Lykkemark, Simon

    2012-01-01

    Directed evolution of antibodies through phage display is a powerful technique for producing binders of various biological targets. One of the recent innovations in the fi eld is the domain antibody, an antibody consisting only of a single variable domain. These anti bodies can be obtained either...... through immunisation of sharks or camels, or alternatively from recombinant libraries1. The domain antibodies have certain advantages, both pharmacologically and technically. Here we report the construction of a semi-synthetic and highly modular antibody library, based on a human framework (V3-23/D47......). The antibody scaffold has been codon optimised to improve expression, and the CDR’s have been created using trinucleotide synthesis. These methods give a strict control over the randomisations, and the ability to design a library with minimal aggregation propensity. To facilitate further manipulation, unique...

  1. Use of phage display to identify novel mineralocorticoid receptor-interacting proteins.

    Science.gov (United States)

    Yang, Jun; Fuller, Peter J; Morgan, James; Shibata, Hirotaka; McDonnell, Donald P; Clyne, Colin D; Young, Morag J

    2014-09-01

    The mineralocorticoid receptor (MR) plays a central role in salt and water homeostasis via the kidney; however, inappropriate activation of the MR in the heart can lead to heart failure. A selective MR modulator that antagonizes MR signaling in the heart but not the kidney would provide the cardiovascular protection of current MR antagonists but allow for normal electrolyte balance. The development of such a pharmaceutical requires an understanding of coregulators and their tissue-selective interactions with the MR, which is currently limited by the small repertoire of MR coregulators described in the literature. To identify potential novel MR coregulators, we used T7 phage display to screen tissue-selective cDNA libraries for MR-interacting proteins. Thirty MR binding peptides were identified, from which three were chosen for further characterization based on their nuclear localization and their interaction with other MR-interacting proteins or, in the case of x-ray repair cross-complementing protein 6, its known status as an androgen receptor coregulator. Eukaryotic elongation factor 1A1, structure-specific recognition protein 1, and x-ray repair cross-complementing protein 6 modulated MR-mediated transcription in a ligand-, cell- and/or promoter-specific manner and colocalized with the MR upon agonist treatment when imaged using immunofluorescence microscopy. These results highlight the utility of phage display for rapid and sensitive screening of MR binding proteins and suggest that eukaryotic elongation factor 1A1, structure-specific recognition protein 1, and x-ray repair cross-complementing protein 6 may be potential MR coactivators whose activity is dependent on the ligand, cellular context, and target gene promoter.

  2. Selective inhibitors of digestive enzymes from Aedes aegypti larvae identified by phage display.

    Science.gov (United States)

    Soares, Tatiane Sanches; Soares Torquato, Ricardo Jose; Alves Lemos, Francisco Jose; Tanaka, Aparecida Sadae

    2013-01-01

    Dengue is a serious disease transmitted by the mosquito Aedes aegypti during blood meal feeding. It is estimated that the dengue virus is transmitted to millions of individuals each year in tropical and subtropical areas. Dengue control strategies have been based on controlling the vector, Ae. aegypti, using insecticide, but the emergence of resistance poses new challenges. The aim of this study was the identification of specific protease inhibitors of the digestive enzymes from Ae. aegypti larvae, which may serve as a prospective alternative biocontrol method. High affinity protein inhibitors were selected by all of the digestive serine proteases of the 4th instar larval midgut, and the specificity of these inhibitors was characterized. These inhibitors were obtained from a phage library displaying variants of HiTI, a trypsin inhibitor from Haematobia irritans, that are mutated in the reactive loop (P1-P4'). Based on the selected amino acid sequence pattern, seven HiTI inhibitor variants were cloned, expressed and purified. The results indicate that the HiTI variants named T6 (RGGAV) and T128 (WNEGL) were selected by larval trypsin-like (IC(50) of 1.1 nM) and chymotrypsin-like enzymes (IC(50) of 11.6 nM), respectively. The variants T23 (LLGGL) and T149 (GGVWR) inhibited both larval chymotrypsin-like (IC(50) of 4.2 nM and 29.0 nM, respectively) and elastase-like enzymes (IC(50) of 1.2 nM for both). Specific inhibitors were successfully obtained for the digestive enzymes of Ae. aegypti larvae by phage display. Our data also strongly suggest the presence of elastase-like enzymes in Ae. aegypti larvae. The HiTI variants T6 and T23 are good candidates for the development as a larvicide to control the vector.

  3. Variable domain antibodies specific for viral hemorrhagic septicemia virus (VHSV) selected from a randomized IgNAR phage display library.

    Science.gov (United States)

    Ohtani, Maki; Hikima, Jun-ichi; Jung, Tae-Sung; Kondo, Hidehiro; Hirono, Ikuo; Takeyama, Haruko; Aoki, Takashi

    2013-02-01

    Phage display libraries are used to screen for nucleotide sequences that encode immunoglobulin variable (V) regions that are specific for a target antigen. We previously constructed an immunoglobulin new antigen receptor (IgNAR) phage display library. Here we used this library to obtain an IgNAR V region that is specific for viral hemorrhagic septicemia virus (VHSV). A phage clone (clone 653) was found to be specific for VHSV by the biopanning method. The V region of clone 653 was used to construct a 6 × His tagged recombinant IgNAR-653 V protein (rIgNAR-653) using the Escherichia coli pET system. The rIgNAR-653 protein bound specifically to VHSV, confirming its activity.

  4. Rapid development of new protein biosensors utilizing peptides obtained via phage display.

    Directory of Open Access Journals (Sweden)

    Jun Wu

    Full Text Available There is a consistent demand for new biosensors for the detection of protein targets, and a systematic method for the rapid development of new sensors is needed. Here we present a platform where short unstructured peptides that bind to a desired target are selected using M13 phage display. The selected peptides are then chemically synthesized and immobilized on gold, allowing for detection of the target using electrochemical techniques such as electrochemical impedance spectroscopy (EIS. A quartz crystal microbalance (QCM is also used as a diagnostic tool during biosensor development. We demonstrate the utility of this approach by creating a novel peptide-based electrochemical biosensor for the enzyme alanine aminotransferase (ALT, a well-known biomarker of hepatotoxicity. Biopanning of the M13 phage display library over immobilized ALT, led to the rapid identification of a new peptide (ALT5-8 with an amino acid sequence of WHWRNPDFWYLK. Phage particles expressing this peptide exhibited nanomolar affinity for immobilized ALT (K(d,app = 85±20 nM. The newly identified ALT5-8 peptide was then chemically synthesized with a C-terminal cysteine for gold immobilization. The performance of the gold-immobilized peptides was studied with cyclic voltammetry (CV, QCM, and EIS. Using QCM, the sensitivity for ALT detection was 8.9±0.9 Hz/(µg/mL and the limit of detection (LOD was 60 ng/mL. Using EIS measurements, the sensitivity was 142±12 impedance percentage change %/(µg/mL and the LOD was 92 ng/mL. In both cases, the LOD was below the typical concentration of ALT in human blood. Although both QCM and EIS produced similar LODs, EIS is preferable due to a larger linear dynamic range. Using QCM, the immobilized peptide exhibited a nanomolar dissociation constant for ALT (K(d = 20.1±0.6 nM. These results demonstrate a simple and rapid platform for developing and assessing the performance of sensitive, peptide-based biosensors for new protein

  5. Flat panel display - Impurity doping technology for flat panel displays

    Energy Technology Data Exchange (ETDEWEB)

    Suzuki, Toshiharu [Advanced Technology Planning, Sumitomo Eaton Nova Corporation, SBS Tower 9F, 10-1, Yoga 4-chome, Setagaya-ku, 158-0097 Tokyo (Japan)]. E-mail: suzuki_tsh@senova.co.jp

    2005-08-01

    Features of the flat panel displays (FPDs) such as liquid crystal display (LCD) and organic light emitting diode (OLED) display, etc. using low temperature poly-Si (LTPS) thin film transistors (TFTs) are briefly reviewed comparing with other FPDs. The requirements for fabricating TFTs used for high performance FPDs and system on glass (SoG) are addressed. This paper focuses on the impurity doping technology, which is one of the key technologies together with crystallization by laser annealing, formation of high quality gate insulator and gate-insulator/poly-Si interface. The issues to be solved in impurity doping technology for state of the art and future TFTs are clarified.

  6. Phage display biopanning and isolation of target-unrelated peptides: in search of nonspecific binders hidden in a combinatorial library.

    Science.gov (United States)

    Bakhshinejad, Babak; Zade, Hesam Motaleb; Shekarabi, Hosna Sadat Zahed; Neman, Sara

    2016-12-01

    Phage display is known as a powerful methodology for the identification of targeting ligands that specifically bind to a variety of targets. The high-throughput screening of phage display combinatorial peptide libraries is performed through the affinity selection method of biopanning. Although phage display selection has proven very successful in the discovery of numerous high-affinity target-binding peptides with potential application in drug discovery and delivery, the enrichment of false-positive target-unrelated peptides (TUPs) without any actual affinity towards the target remains a major problem of library screening. Selection-related TUPs may emerge because of binding to the components of the screening system rather than the target. Propagation-related TUPs may arise as a result of faster growth rate of some phage clones enabling them to outcompete slow-propagating clones. Amplification of the library between rounds of biopanning makes a significant contribution to the selection of phage clones with propagation advantage. Distinguishing nonspecific TUPs from true target binders is of particular importance for the translation of biopanning findings from basic research to clinical applications. Different experimental and in silico approaches are applied to assess the specificity of phage display-derived peptides towards the target. Bioinformatic tools are playing a rapidly growing role in the analysis of biopanning data and identification of target-irrelevant TUPs. Recent progress in the introduction of efficient strategies for TUP detection holds enormous promise for the discovery of clinically relevant cell- and tissue-homing peptides and paves the way for the development of novel targeted diagnostic and therapeutic platforms in pharmaceutical areas.

  7. Functional characterization of a monoclonal antibody epitope using a lambda phage display-deep sequencing platform

    Science.gov (United States)

    Domina, Maria; Lanza Cariccio, Veronica; Benfatto, Salvatore; Venza, Mario; Venza, Isabella; Borgogni, Erica; Castellino, Flora; Midiri, Angelina; Galbo, Roberta; Romeo, Letizia; Biondo, Carmelo; Masignani, Vega; Teti, Giuseppe; Felici, Franco; Beninati, Concetta

    2016-01-01

    We have recently described a method, named PROFILER, for the identification of antigenic regions preferentially targeted by polyclonal antibody responses after vaccination. To test the ability of the technique to provide insights into the functional properties of monoclonal antibody (mAb) epitopes, we used here a well-characterized epitope of meningococcal factor H binding protein (fHbp), which is recognized by mAb 12C1. An fHbp library, engineered on a lambda phage vector enabling surface expression of polypeptides of widely different length, was subjected to massive parallel sequencing of the phage inserts after affinity selection with the 12C1 mAb. We detected dozens of unique antibody-selected sequences, the most enriched of which (designated as FrC) could largely recapitulate the ability of fHbp to bind mAb 12C1. Computational analysis of the cumulative enrichment of single amino acids in the antibody-selected fragments identified two overrepresented stretches of residues (H248-K254 and S140-G154), whose presence was subsequently found to be required for binding of FrC to mAb 12C1. Collectively, these results suggest that the PROFILER technology can rapidly and reliably identify, in the context of complex conformational epitopes, discrete “hot spots” with a crucial role in antigen-antibody interactions, thereby providing useful clues for the functional characterization of the epitope. PMID:27530334

  8. Selection of Immunogenic Peptide Mimics of Male Worm Origin of Schistosoma Japonicum using Phage Display Peptide Library

    Institute of Scientific and Technical Information of China (English)

    陈欲晓; 易新元; 曾宪芳; 王林纤; 唐连飞; 章洁; McreynoldsLarry

    2004-01-01

    To select the immunogenic short peptide mimics of male worm origin of Schistosoma japonicum (Sj) and to explore their protection effect against schistosomiasis in mice, the random phage display peptide hbrary of 12 - mer was screened with IgG to soluble male worm antigen of Sj, and the specific positive clones selected through three rounds of screenings were detected by Dot-ELISA, and then injected subcutaneously into mice for vaccination and protection assessment against Sj. It was found that 18 randomly picked phage displayed clones all showed definite antigenicity with various intensities. The pooled phages displayed clones could induce production of specific antibodies and cause 31.72% of worm reduction rate and 51.54 % of egg reduction rate in mice, revealing a significant difference ( P < 0. 001 ) in comparison with those of the controls. It concludes that the short peptide mimics of male worm origin of Sj obtained by affinity screening phage display ptide library can elicit partial protection against this pathogen.

  9. Phage display used for gene cloning of human recombinant antibody against the erythrocyte surface antigen, rhesus D

    DEFF Research Database (Denmark)

    Dziegiel, M; Nielsen, L K; Andersen, P S;

    1995-01-01

    A novel phage display system has been developed for PCR amplification and cloning of the Fab fragments of human immunoglobulin genes. Using this system, we have cloned an antibody from a mouse-human hybridoma cell line directed against the erythrocyte antigen rhesus D. Intact erythrocytes were used...

  10. Phage display used for gene cloning of human recombinant antibody against the erythrocyte surface antigen, rhesus D

    DEFF Research Database (Denmark)

    Dziegiel, M; Nielsen, L K; Andersen, P S

    1995-01-01

    A novel phage display system has been developed for PCR amplification and cloning of the Fab fragments of human immunoglobulin genes. Using this system, we have cloned an antibody from a mouse-human hybridoma cell line directed against the erythrocyte antigen rhesus D. Intact erythrocytes were us...

  11. Glycoarrays with engineered phages displaying structurally diverse oligosaccharides enable high-throughput detection of glycan-protein interactions

    Science.gov (United States)

    Çelik, Eda; Ollis, Anne A.; Lasanajak, Yi; Fisher, Adam C.; Gür, Göksu; Smith, David F.; DeLisa, Matthew P.

    2014-01-01

    Glycan microarrays have become a powerful platform to investigate the interactions of carbohydrates with a variety of biomolecules. However, the number and diversity of glycans available for use in such arrays represents a key bottleneck in glycan array fabrication. To address this challenge, we describe a novel glycan array platform based on surface patterning of engineered glycophages that display unique carbohydrate epitopes. Specifically, we show that glycophages are compatible with surface immobilization procedures and that phage-displayed oligosaccharides retain the ability to be recognized by different glycan-binding proteins (e.g., antibodies, lectins) after immobilization. A key advantage of glycophage arrays is that large quantities of glycophages can be produced biosynthetically from recombinant bacteria and isolated directly from bacterial supernatants without laborious purification steps. Taken together, the glycophage array technology described here should help to expand the diversity of glycan libraries and provide a complement to the existing toolkit for high-throughput analysis of glycan-protein interactions. PMID:25263089

  12. Enrichment of an in vivo phage display repertoire by subtraction for easy identification of pathology biomarkers

    Directory of Open Access Journals (Sweden)

    karina Vargas Sanchez

    2015-03-01

    Conclusion. This physical subtraction discarded from a complex repertoire the non-specific selected ligands. STRATEGY 1 Three rounds of in vivo phage peptide selection in EAE female Lewis rats ("EAE repertoire" vs controls ("HEALTHY repertoire". 2 DNA subtraction of the most common sequences between «HEALTHY» and «EAE» phage repertoires to obtain a third EAE specific «SUBTRACTION » phage repertoire. 3 Massive sequencing of the three repertoires and bioinformatic analysis to identify the peptides sequences with high EAE specificity. 4 Biological tests of potential EAE specific phage clones with CNS tissues from EAE and Healthy control rats. 5 Biological tests of the EAE specific peptide and phage clones on the BBB in vitro model (hCMEC/D3 cells under inflammatory conditions (IL-1β stimulation. 6 Target separation and identification by cross-link between the selected phage clones and hMEC/D3 endothelial cells targets under IL-1β stimulation vs controls.

  13. Phage display used for gene cloning of human recombinant antibody against the erythrocyte surface antigen, rhesus D

    DEFF Research Database (Denmark)

    Dziegiel, Morten Hanefeld; Nielsen, L K; Andersen, P S

    1995-01-01

    A novel phage display system has been developed for PCR amplification and cloning of the Fab fragments of human immunoglobulin genes. Using this system, we have cloned an antibody from a mouse-human hybridoma cell line directed against the erythrocyte antigen rhesus D. Intact erythrocytes were used...... for absorption of the Fab phages. Soluble Fab fragments produced from the cloned material showed identical performance to the parental antibody in agglutination assays. Gel filtration confirmed that the Fab fragment consists of a kappa-Fd heterodimer. The successful use of intact cells for selection of specific...... Fab phages demonstrates that it is possible to by-pass purification of the antigen of interest. Comparison with published germline sequences demonstrated that the immunoglobulin coding regions had the highest homology to the VH 1.9III and V kappa Hum kappa v325 germline genes, respectively....

  14. Phage display-derived inhibitor of the essential cell wall biosynthesis enzyme MurF

    Directory of Open Access Journals (Sweden)

    Blewett Ann

    2008-12-01

    Full Text Available Abstract Background To develop antibacterial agents having novel modes of action against bacterial cell wall biosynthesis, we targeted the essential MurF enzyme of the antibiotic resistant pathogen Pseudomonas aeruginosa. MurF catalyzes the formation of a peptide bond between D-Alanyl-D-Alanine (D-Ala-D-Ala and the cell wall precursor uridine 5'-diphosphoryl N-acetylmuramoyl-L-alanyl-D-glutamyl-meso-diaminopimelic acid (UDP-MurNAc-Ala-Glu-meso-A2pm with the concomitant hydrolysis of ATP to ADP and inorganic phosphate, yielding UDP-N-acetylmuramyl-pentapeptide. As MurF acts on a dipeptide, we exploited a phage display approach to identify peptide ligands having high binding affinities for the enzyme. Results Screening of a phage display 12-mer library using purified P. aeruginosa MurF yielded to the identification of the MurFp1 peptide. The MurF substrate UDP-MurNAc-Ala-Glumeso-A2pm was synthesized and used to develop a sensitive spectrophotometric assay to quantify MurF kinetics and inhibition. MurFp1 acted as a weak, time-dependent inhibitor of MurF activity but was a potent inhibitor when MurF was pre-incubated with UDP-MurNAc-Ala-Glu-meso-A2pm or ATP. In contrast, adding the substrate D-Ala-D-Ala during the pre-incubation nullified the inhibition. The IC50 value of MurFp1 was evaluated at 250 μM, and the Ki was established at 420 μM with respect to the mixed type of inhibition against D-Ala-D-Ala. Conclusion MurFp1 exerts its inhibitory action by interfering with the utilization of D-Ala-D-Ala by the MurF amide ligase enzyme. We propose that MurFp1 exploits UDP-MurNAc-Ala-Glu-meso-A2pm-induced structural changes for better interaction with the enzyme. We present the first peptide inhibitor of MurF, an enzyme that should be exploited as a target for antimicrobial drug development.

  15. Immunodiagnosis of human neurocysticercosis using a synthetic peptide selected by phage-display.

    Science.gov (United States)

    Hell, R C R; Amim, P; de Andrade, H M; de Avila, R A M; Felicori, L; Oliveira, A G; Oliveira, C A; Nascimento, E; Tavares, C A P; Granier, C; Chávez-Olórtegui, C

    2009-04-01

    The usefulness of a synthetic peptide in the serodiagnosis of Taenia solium human neurocysticercosis (NC) has been evaluated. Phage-displayed peptides were screened with human antibodies to scolex protein antigen from cysticercus cellulosae (SPACc). One clone was found to interact specifically with anti-SPACc IgGs. The corresponding synthetic peptide was found to be recognized in ELISA by NC patient's sera. The study was carried out with sera from 28 confirmed NC patients, 13 control sera and 73 sera from patients suffering from other infectious diseases. A 93% sensibility and a 94.3% specificity was achieved. Figures of 89% and 31.4% of sensibility and specificity were obtained in a SPACc-based ELISA. Immunoblotting of SPACc with anti-peptide antibodies revealed a single band of approximately 45 kDa in 1D and four 45 kDa isoforms in 2D-gel electrophoresis. A strong and specific immunostaining in the fibers beneath the suckers, at the base of the rostellum, and in the tissue surrounding the scolex of cysticerci was observed by immunomicroscopy. Our results show that a peptide-based immunodiagnostic of neurocisticercosis can be envisioned.

  16. Identification of a novel skin penetration enhancement peptide by phage display peptide library screening.

    Science.gov (United States)

    Kumar, Sunny; Sahdev, Preety; Perumal, Omathanu; Tummala, Hemachand

    2012-05-07

    Skin is an important site for local or systemic application of drugs. However, a majority of drugs have poor permeability through the skin's topmost layer, stratum corneum (SC). The aim of this study was to identify safe and smaller peptides that could enhance the skin penetration of drug molecules. By screening phage display peptide library, we have identified a T2 peptide (LVGVFH), which enhanced the penetration of bacteriophages (~800 nm long bacterial viruses) across porcine and mouse skin. Pretreating the skin with synthetic T2 peptide at pH 4.5 resulted in significant penetration enhancement of hydrophilic drug 5-fluorouracil (5-FU) across skin. FTIR spectroscopy showed that the T2 peptide interacted with skin lipids to enhance the skin penetration. Pretreating the skin with T2 peptide enhanced the partitioning of small molecules with different lipophilicities (5-FU, fluorescein isothiocyanate, and rhodamine 123 hydrochloride) into skin. Fluorescence studies showed that T2 peptide enhanced the diffusion of these molecules into intercellular lipids of SC and thus enhanced the penetration into the skin. Histidine at the c-terminus of T2 peptide was identified to be critical for the skin penetration enhancement. T2 peptide interacted with skin lipids to cause skin penetration enhancement. The study identified a novel, safe, and noninvasive peptide to improve the skin penetration of drugs without chemical conjugation.

  17. Epitope Mapping of Metuximab on CD147 Using Phage Display and Molecular Docking

    Directory of Open Access Journals (Sweden)

    Bifang He

    2013-01-01

    Full Text Available Metuximab is the generic name of Licartin, a new drug for radioimmunotherapy of hepatocellular carcinoma. Although it is known to be a mouse monoclonal antibody against CD147, the complete epitope mediating the binding of metuximab to CD147 remains unknown. We panned the Ph.D.-12 phage display peptide library against metuximab and got six mimotopes. The following bioinformatics analysis based on mimotopes suggested that metuximab recognizes a conformational epitope composed of more than 20 residues. The residues of its epitope may include T28, V30, K36, L38, K57, F74, D77, S78, D79, D80, Q81, G83, S86, N98, Q100, L101, H102, G103, P104, V131, P132, and K191. The homology modeling of metuximab and the docking of CD147 to metuximab were also performed. Based on the top one docking model, the epitope was predicted to contain 28 residues: AGTVFTTV (23–30, I37, D45, E84, V88, EPMGTANIQLH (92–102, VPP (131–133, Q164, and K191. Almost half of the residues predicted on the basis of mimotope analysis also appear in the docking result, indicating that both results are reliable. As the predicted epitopes of metuximab largely overlap with interfaces of CD147-CD147 interactions, a structural mechanism of metuximab is proposed as blocking the formation of CD147 dimer.

  18. Isolation and characterization of anti ROR1 single chain fragment variable antibodies using phage display technique.

    Science.gov (United States)

    Aghebati-Maleki, Leili; Younesi, Vahid; Jadidi-Niaragh, Farhad; Baradaran, Behzad; Majidi, Jafar; Yousefi, Mehdi

    2017-01-01

    Receptor tyrosine kinase-like orphan receptor (ROR1) belongs to one of the families of receptor tyrosine kinases (RTKs). RTKs are involved in the various physiologic cellular functions including proliferation, migration, survival, signaling and differentiation. Several RTKs are deregulated in various cancers implying the targeting potential of these molecules in cancer therapy. ROR1 has recently been shown to be expressed in various types of cancer cells but not in normal adult cells. Hence a molecular inhibitor of extracellular domain of ROR1 that inhibits ROR1-cell surface interaction is of great therapeutic importance. In an attempt to develop molecular inhibitors of ROR1, we screened single chain variable fragment (scFv) phage display libraries, Tomlinson I + J, against one specific synthetic oligopeptide from extracellular domain of ROR1 and selected scFvs were characterized using various immunological techniques. Several ROR1 specific scFvs were selected following five rounds of panning procedure. The scFvs showed specific binding to ROR1 using immunological techniques. Our results demonstrate successful isolation and characterization of specific ROR1 scFvs that may have great therapeutic potential in cancer immunotherapy.

  19. Construction of white spot syndrome virus (WSSV) whole genome phage display library

    Institute of Scientific and Technical Information of China (English)

    ZHU Yanbing; YANG Feng

    2007-01-01

    A rebuilt vector pCANTAB 5 EE was obtained by inserting a 34 bp double-stranded oligonucleotide which contained a EcoRV recognition sequence into pCANTAB 5 E. White spot syndrome virus (WSSV) genome DNA was fragmented by sonication to isolate fragments mainly in the range of 0.8 ~2.0 kb, then the fragments were blunt-ended with T4 DNA polymerase and cloned into the EcoRV site of pCANTAB 5 EE. The primary recombinant clone of the library was 3.0 × 105.Colony PCR of random selected recombinants showed that the size of the inserts was 0.12 ~ 1.77 kb. After the whole library recombinant phages infected Escherichia coli HB2151 cells, the extracellular and periplasmic extracts were dropped on PVDF membranes to perform dot blot, using polyclonal mouse anti-VP24 serum,anti-WSV026 serum,anti-WSV063 serum,anti-WSV069 serum,anti-WSV112 serum, anti WSV238 serum,anti-WSV303 serum and anti-VP26 serum as the primary antibody, respectively. The results showed that the display library could express the viral proteins.

  20. Structural guided scaffold phage display libraries as a source of bio-therapeutics.

    Directory of Open Access Journals (Sweden)

    Y K Stella Man

    Full Text Available We have developed a structurally-guided scaffold phage display strategy for identification of ligand mimetic bio-therapeutics. As a proof of concept we used the ligand of integrin αvβ6, a tumour cell surface receptor and a major new target for imaging and therapy of many types of solid cancer. NMR structure analysis showed that RGD-helix structures are optimal for αvβ6 ligand-interaction, so we designed novel algorithms to generate human single chain fragment variable (scFv libraries with synthetic VH-CDR3 encoding RGD-helix hairpins with helices of differing pitch, length and amino acid composition. Study of the lead scFv clones D25scFv and D34scFv and their corresponding VH-CDR3 derived peptides, D25p and D34p, demonstrated: specific binding to recombinant and cellular αvβ6; inhibition of αvβ6-dependent cell and ligand adhesion, αvβ6-dependent cell internalisation; and selective retention by αvβ6-expressing, but not αvβ6-negative, human xenografts. NMR analysis established that both the D25p and D34p retained RGD-helix structures confirming the success of the algorithm. In conclusion, scFv libraries can be engineered based on ligand structural motifs to increase the likelihood of developing powerful bio-therapeutics.

  1. Epitope mapping of metuximab on CD147 using phage display and molecular docking.

    Science.gov (United States)

    He, Bifang; Mao, Canquan; Ru, Beibei; Han, Hesong; Zhou, Peng; Huang, Jian

    2013-01-01

    Metuximab is the generic name of Licartin, a new drug for radioimmunotherapy of hepatocellular carcinoma. Although it is known to be a mouse monoclonal antibody against CD147, the complete epitope mediating the binding of metuximab to CD147 remains unknown. We panned the Ph.D.-12 phage display peptide library against metuximab and got six mimotopes. The following bioinformatics analysis based on mimotopes suggested that metuximab recognizes a conformational epitope composed of more than 20 residues. The residues of its epitope may include T28, V30, K36, L38, K57, F74, D77, S78, D79, D80, Q81, G83, S86, N98, Q100, L101, H102, G103, P104, V131, P132, and K191. The homology modeling of metuximab and the docking of CD147 to metuximab were also performed. Based on the top one docking model, the epitope was predicted to contain 28 residues: AGTVFTTV (23-30), I37, D45, E84, V88, EPMGTANIQLH (92-102), VPP (131-133), Q164, and K191. Almost half of the residues predicted on the basis of mimotope analysis also appear in the docking result, indicating that both results are reliable. As the predicted epitopes of metuximab largely overlap with interfaces of CD147-CD147 interactions, a structural mechanism of metuximab is proposed as blocking the formation of CD147 dimer.

  2. Emerging Technologies of Liquid Crystal Displays

    Institute of Scientific and Technical Information of China (English)

    Sin-Doo Lee; Chang-Jae Yu; Jae-Hong Park; Min-Sik Jung

    2003-01-01

    The general features and the emerging technologies of liquid crystal displays are described from the viewpoints of wide viewing and fast response technologies. The device applications of liquid crystals for optical communications are also described.

  3. Anti-idiotypic VHH phage display-mediated immuno-PCR for ultrasensitive determination of mycotoxin zearalenone in cereals.

    Science.gov (United States)

    Wang, Xianxian; He, Qinghua; Xu, Yang; Liu, Xing; Shu, Mei; Tu, Zhui; Li, Yanping; Wang, Wei; Cao, Dongmei

    2016-01-15

    Immunoassay is frequently used to analyze mycotoxin contamination. However, the introduction of mycotoxins or their conjugates in conventional immunoassay threatens the safety of individuals and the environment. The variable domain of heavy-chain antibodies (VHHs) can be used as alternative compounds to produce anti-idiotypic antibodies, which work as non-toxic surrogate reagents in immunoassay. In this work, anti-zearalenone (ZEN) monoclonal antibody (mAb) was used as the target for biopanning anti-idiotypic VHH from a naïve alpaca VHH phage display library. After four panning cycles, one anti-idiotypic VHH phage clone (Z1) was isolated and the Z1 based phage ELISA for ZEN showed a half inhibitory concentration (IC50) of 0.25±0.02ng/mL, a linear range of 0.11-0.55ng/mL, and a limit of detection (LOD) of 0.08ng/mL. Furthermore, the phage particles of Z1 were also applied to immuno-polymerase chain reaction (PD-IPCR), which supplied both the detection antigens and deoxyribonucleic acid (DNA) templates. Compared with that of phage ELISA, the LOD of Z1 based PD-IPCR was 12-fold improved, with a detection limit of 6.5pg/mL and a linear range of 0.01-100ng/mL. The proposed method was then validated with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results showed the reliability of PD-IPCR for the determination of ZEN in cereal samples. The use of anti-idiotypic VHH phage as non-toxic surrogate and signal-amplification function of PCR make it a promising method for actual ZEN analysis in cereals. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Potential of Peptides as Inhibitors and Mimotopes: Selection of Carbohydrate-Mimetic Peptides from Phage Display Libraries

    Directory of Open Access Journals (Sweden)

    Teruhiko Matsubara

    2012-01-01

    Full Text Available Glycoconjugates play various roles in biological processes. In particular, oligosaccharides on the surface of animal cells are involved in virus infection and cell-cell communication. Inhibitors of carbohydrate-protein interactions are potential antiviral drugs. Several anti-influenza drugs such as oseltamivir and zanamivir are derivatives of sialic acid, which inhibits neuraminidase. However, it is very difficult to prepare a diverse range of sugar derivatives by chemical synthesis or by the isolation of natural products. In addition, the pathogenic capsular polysaccharides of bacteria are carbohydrate antigens, for which a safe and efficacious method of vaccination is required. Phage-display technology has been improved to enable the identification of peptides that bind to carbohydrate-binding proteins, such as lectins and antibodies, from a large repertoire of peptide sequences. These peptides are known as “carbohydrate-mimetic peptides (CMPs” because they mimic carbohydrate structures. Compared to carbohydrate derivatives, it is easy to prepare mono- and multivalent peptides and then to modify them to create various derivatives. Such mimetic peptides are available as peptide inhibitors of carbohydrate-protein interactions and peptide mimotopes that are conjugated with adjuvant for vaccination.

  5. The T7-related Pseudomonas putida phage φ15 displays virion-associated biofilm degradation properties.

    Directory of Open Access Journals (Sweden)

    Anneleen Cornelissen

    Full Text Available Formation of a protected biofilm environment is recognized as one of the major causes of the increasing antibiotic resistance development and emphasizes the need to develop alternative antibacterial strategies, like phage therapy. This study investigates the in vitro degradation of single-species Pseudomonas putida biofilms, PpG1 and RD5PR2, by the novel phage ϕ15, a 'T7-like virus' with a virion-associated exopolysaccharide (EPS depolymerase. Phage ϕ15 forms plaques surrounded by growing opaque halo zones, indicative for EPS degradation, on seven out of 53 P. putida strains. The absence of haloes on infection resistant strains suggests that the EPS probably act as a primary bacterial receptor for phage infection. Independent of bacterial strain or biofilm age, a time and dose dependent response of ϕ15-mediated biofilm degradation was observed with generally a maximum biofilm degradation 8 h after addition of the higher phage doses (10(4 and 10(6 pfu and resistance development after 24 h. Biofilm age, an in vivo very variable parameter, reduced markedly phage-mediated degradation of PpG1 biofilms, while degradation of RD5PR2 biofilms and ϕ15 amplification were unaffected. Killing of the planktonic culture occurred in parallel with but was always more pronounced than biofilm degradation, accentuating the need for evaluating phages for therapeutic purposes in biofilm conditions. EPS degrading activity of recombinantly expressed viral tail spike was confirmed by capsule staining. These data suggests that the addition of high initial titers of specifically selected phages with a proper EPS depolymerase are crucial criteria in the development of phage therapy.

  6. Identification of the specificity of isolated phage display single-chain antibodies using yeast two-hybrid screens

    DEFF Research Database (Denmark)

    Rasmussen, Nicolaj; Ditzel, Henrik

    2009-01-01

    A method is described for the identification of the antigen recognised by an scFv isolated from an antibody phage display library using selection against a complex mixture of proteins (e.g. intact cells, purified cell surface membranes, and tissue sections). The method takes advantage of a yeast ...... two-hybrid system that additionally allows for reorganization of post-translational modifications to the bait and target proteins. This technique is therefore especially useful for identifying surface-expressed antigens....

  7. Phage display in vivo selection and its application%噬菌体展示体内筛选技术及其应用进展

    Institute of Scientific and Technical Information of China (English)

    俞杨; 王自正

    2008-01-01

    Phage display technology is a kind of method which display peptides or proteins of high diversity on the surface of phage coat pmteins.This technology establish a linkage between genotype and phenotype,which makes functional screen of peptides or proteins convenient.Peptides or proteins screened from phage display library have high specificity and strong affinity,and play an important roles in basic research and clinical diagnosis and therapy.With the development of phage display,it has been used in the in vivo selection.It has characteristics of high throughput selection so that it can reveal molecular profiles and the differences of molecular profiles expressed on the vascular surface of different organs.Studies of in vivo selection can provide experimental evidences for the targeting diagnosis and therapy.%噬菌体展示技术是将高度多样性的多肽或蛋白展示于噬菌体衣壳蛋白表面的一项技术.这一技术将展示分子的基因型和表型联系在一起,极大地方便了多肽或蛋白分子的功能性筛选.从噬菌体展示文库中筛选出的多肽或蛋白具有高度的特异性和极强的亲和力,在生物医学基础研究和临床诊断治疗中具有重要的应用价值.随着噬菌体展示技术的不断发展,已将其用于活体的体内筛选.体内筛选技术发挥了高通量筛选的特点,能够在活体水平研究血管表面的分子表达状况,分析不同器官血管表面分子的表达差异,从而为临床靶向性诊断和治疗提供实验依据.

  8. Modern Display Technologies and Applications

    Science.gov (United States)

    1982-01-01

    conventional tubes, LSI circuitry offers the possibility of correcting some of the deficiencies in electron-optic perform- ance and may lead to acceptable...certain ceramic materials such as PLZT (lead lanthanum zirconate titanate) can be utilized for display applications. PLZT is transparent in the visible...consuming power (3.8.12). 3.8.4.2 State of development, Magnetic particles have been made of polyethylene with powdered Strontium ferrite as a filler

  9. New ultraportable display technology and applications

    Science.gov (United States)

    Alvelda, Phillip; Lewis, Nancy D.

    1998-08-01

    MicroDisplay devices are based on a combination of technologies rooted in the extreme integration capability of conventionally fabricated CMOS active-matrix liquid crystal display substrates. Customized diffraction grating and optical distortion correction technology for lens-system compensation allow the elimination of many lenses and systems-level components. The MicroDisplay Corporation's miniature integrated information display technology is rapidly leading to many new defense and commercial applications. There are no moving parts in MicroDisplay substrates, and the fabrication of the color generating gratings, already part of the CMOS circuit fabrication process, is effectively cost and manufacturing process-free. The entire suite of the MicroDisplay Corporation's technologies was devised to create a line of application- specific integrated circuit single-chip display systems with integrated computing, memory, and communication circuitry. Next-generation portable communication, computer, and consumer electronic devices such as truly portable monitor and TV projectors, eyeglass and head mounted displays, pagers and Personal Communication Services hand-sets, and wristwatch-mounted video phones are among the may target commercial markets for MicroDisplay technology. Defense applications range from Maintenance and Repair support, to night-vision systems, to portable projectors for mobile command and control centers.

  10. A resistin binding peptide selected by phage display inhibits 3T3-L1 preadipocyte differentiation

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Background Resistin, a newly discovered cysteine-rich hormone secreted mainly by adipose tissues, has been proposed to form a biochemical link between obesity and type 2 diabetes. However, the resistin receptor has not yet been identified. This study aimed to identify resistin binding proteins/receptor.Methods Three cDNA fragments with the same 11 bp 5' sequence were found by screening a cDNA phage display library of rat multiple tissues. As the reading frames of the same 11 bp 5' sequence were interrupted by a TGA stop codon, plaque lift assay was consequently used to prove the readthrough phenomenon. The stop codon in the same 11 bp 5' sequence was replaced by tryptophan, and the binding activity of the coded peptide [AWIL, which was designated as resistin binding peptide (RBP)] with resistin was identified by the confocal microscopy technique and the affinity chromatography experiment. pDual GC-resistin and pDual GC-resistin binding peptide were co-transfected into 3T3-L1 cells to confirm the function of resistin binding peptide.Results Three cDNA fragments with the same 11 bp 5' sequence were found. The TGA stop codon in reading frames of the same 11 bp 5' sequence was proved to be readthroughed. The binding activity of RBP with resistin was consequently identified. The expression of the resistin binding peptide in 3T3-L1 preadipocytes expressing pDual GC-resistin significantly inhibited the adipogenic differentiation.Conclusion RBP could effectively rescue the promoted differentiation of resistin overxepressed 3T3-L1 preadipocyte.

  11. Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library.

    Science.gov (United States)

    Wang, Han; Yu, Rui; Fang, Ting; Yu, Ting; Chi, Xiangyang; Zhang, Xiaopeng; Liu, Shuling; Fu, Ling; Yu, Changming; Chen, Wei

    2016-09-11

    Tetanus neurotoxin (TeNT) produced by Clostridium tetani is one of the most poisonous protein substances. Neutralizing antibodies against TeNT can effectively prevent and cure toxicosis. Using purified Hc fragments of TeNT (TeNT-Hc) as an antigen, three specific neutralizing antibody clones recognizing different epitopes were selected from a human immune scFv antibody phage display library. The three antibodies (2-7G, 2-2D, and S-4-7H) can effectively inhibit the binding between TeNT-Hc and differentiated PC-12 cells in vitro. Moreover, 2-7G inhibited TeNT-Hc binding to the receptor via carbohydrate-binding sites of the W pocket while 2-2D and S-4-7H inhibited binding of the R pocket. Although no single mAb completely protected mice from the toxin, they could both prolong survival when challenged with 20 LD50s (50% of the lethal dose) of TeNT. When used together, the mAbs completely neutralized 1000 LD50s/mg Ab, indicating their high neutralizing potency in vivo. Antibodies recognizing different carbohydrate-binding pockets could have higher synergistic toxin neutralization activities than those that recognize the same pockets. These results could lead to further production of neutralizing antibody drugs against TeNT and indicate that using TeNT-Hc as an antigen for screening human antibodies for TeNT intoxication therapy from human immune antibody library was convenient and effective.

  12. Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library

    Science.gov (United States)

    Wang, Han; Yu, Rui; Fang, Ting; Yu, Ting; Chi, Xiangyang; Zhang, Xiaopeng; Liu, Shuling; Fu, Ling; Yu, Changming; Chen, Wei

    2016-01-01

    Tetanus neurotoxin (TeNT) produced by Clostridium tetani is one of the most poisonous protein substances. Neutralizing antibodies against TeNT can effectively prevent and cure toxicosis. Using purified Hc fragments of TeNT (TeNT-Hc) as an antigen, three specific neutralizing antibody clones recognizing different epitopes were selected from a human immune scFv antibody phage display library. The three antibodies (2-7G, 2-2D, and S-4-7H) can effectively inhibit the binding between TeNT-Hc and differentiated PC-12 cells in vitro. Moreover, 2-7G inhibited TeNT-Hc binding to the receptor via carbohydrate-binding sites of the W pocket while 2-2D and S-4-7H inhibited binding of the R pocket. Although no single mAb completely protected mice from the toxin, they could both prolong survival when challenged with 20 LD50s (50% of the lethal dose) of TeNT. When used together, the mAbs completely neutralized 1000 LD50s/mg Ab, indicating their high neutralizing potency in vivo. Antibodies recognizing different carbohydrate-binding pockets could have higher synergistic toxin neutralization activities than those that recognize the same pockets. These results could lead to further production of neutralizing antibody drugs against TeNT and indicate that using TeNT-Hc as an antigen for screening human antibodies for TeNT intoxication therapy from human immune antibody library was convenient and effective. PMID:27626445

  13. Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library

    Directory of Open Access Journals (Sweden)

    Han Wang

    2016-09-01

    Full Text Available Tetanus neurotoxin (TeNT produced by Clostridium tetani is one of the most poisonous protein substances. Neutralizing antibodies against TeNT can effectively prevent and cure toxicosis. Using purified Hc fragments of TeNT (TeNT-Hc as an antigen, three specific neutralizing antibody clones recognizing different epitopes were selected from a human immune scFv antibody phage display library. The three antibodies (2-7G, 2-2D, and S-4-7H can effectively inhibit the binding between TeNT-Hc and differentiated PC-12 cells in vitro. Moreover, 2-7G inhibited TeNT-Hc binding to the receptor via carbohydrate-binding sites of the W pocket while 2-2D and S-4-7H inhibited binding of the R pocket. Although no single mAb completely protected mice from the toxin, they could both prolong survival when challenged with 20 LD50s (50% of the lethal dose of TeNT. When used together, the mAbs completely neutralized 1000 LD50s/mg Ab, indicating their high neutralizing potency in vivo. Antibodies recognizing different carbohydrate-binding pockets could have higher synergistic toxin neutralization activities than those that recognize the same pockets. These results could lead to further production of neutralizing antibody drugs against TeNT and indicate that using TeNT-Hc as an antigen for screening human antibodies for TeNT intoxication therapy from human immune antibody library was convenient and effective.

  14. DNA immunization combined with scFv phage display identifies antagonistic GCGR specific antibodies and reveals new epitopes on the small extracellular loops.

    Science.gov (United States)

    van der Woning, Bas; De Boeck, Gitte; Blanchetot, Christophe; Bobkov, Vladimir; Klarenbeek, Alex; Saunders, Michael; Waelbroeck, Magali; Laeremans, Toon; Steyaert, Jan; Hultberg, Anna; De Haard, Hans

    2016-01-01

    The identification of functional monoclonal antibodies directed against G-protein coupled receptors (GPCRs) is challenging because of the membrane-embedded topology of these molecules. Here, we report the successful combination of llama DNA immunization with scFv-phage display and selections using virus-like particles (VLP) and the recombinant extracellular domain of the GPCR glucagon receptor (GCGR), resulting in glucagon receptor-specific antagonistic antibodies. By immunizing outbred llamas with plasmid DNA containing the human GCGR gene, we sought to provoke their immune system, which generated a high IgG1 response. Phage selections on VLPs allowed the identification of mAbs against the extracellular loop regions (ECL) of GCGR, in addition to multiple VH families interacting with the extracellular domain (ECD) of GCGR. Identifying mAbs binding to the ECL regions of GCGR is challenging because the large ECD covers the small ECLs in the energetically most favorable 'closed conformation' of GCGR. Comparison of Fab with scFv-phage display demonstrated that the multivalent nature of scFv display is essential for the identification of GCGR specific clones by selections on VLPs because of avid interaction. Ten different VH families that bound 5 different epitopes on the ECD of GCGR were derived from only 2 DNA-immunized llamas. Seven VH families demonstrated interference with glucagon-mediated cAMP increase. This combination of technologies proved applicable in identifying multiple functional binders in the class B GPCR context, suggesting it is a robust approach for tackling difficult membrane proteins.

  15. Phage display selection of tight specific binding variants from a hyperthermostable Sso7d scaffold protein library.

    Science.gov (United States)

    Zhao, Ning; Schmitt, Margaret A; Fisk, John D

    2016-04-01

    Antibodies, the quintessential biological recognition molecules, are not ideal for many applications because of their large size, complex modifications, and thermal and chemical instability. Identifying alternative scaffolds that may be evolved into tight, specific binding molecules with improved physical properties is of increasing interest, particularly for biomedical applications in resource-limited environments. Hyperthermophilic organisms, such as Sulfolobus solfataricus, are an attractive source of highly stable proteins that may serve as starting points for alternative molecular recognition scaffolds. We describe the first application of phage display to identify binding proteins based on the S. solfataricus protein Sso7d scaffold. Sso7d is a small cysteine-free DNA-binding protein (approximately 7 kDa, 63 amino acids), with a melting temperature of nearly 100 °C. Tight-binding Sso7d variants were selected for a diverse set of protein targets from a 10(10) member library, demonstrating the versatility of the scaffold. These Sso7d variants are able to discriminate among closely related human, bovine and rabbit serum albumins. Equilibrium dissociation constants in the nanomolar to low micromolar range were measured via competitive ELISA. Importantly, the Sso7d variants continue to bind their targets in the absence of the phage context. Furthermore, phage-displayed Sso7d variants retain their binding affinity after exposure to temperatures up to 70 °C. Taken together, our results suggest that the Sso7d scaffold will be a complementary addition to the range of non-antibody scaffold proteins that may be utilized in phage display. Variants of hyperthermostable binding proteins have potential applications in diagnostics and therapeutics for environments with extreme conditions of storage and deployment.

  16. Identification of Peptides Inhibiting Adhesion of Monocytes to the Injured Vascular Endothelial Cells through Phage-displaying Screening

    Institute of Scientific and Technical Information of China (English)

    Yu GUO; Jia ZHANG; Ji-Cheng WANG; Feng-Xiang YAN; Bing-Yang ZHU; Hong-Lin HUANG; Duan-Fang LIAO

    2005-01-01

    Using oxidized low-density lipoprotein (LDL)-injured vascular endothelial cells (ECs) as target cells, peptides specifically binding to the injured ECs were screened from a phage-displaying peptide library by using the whole-cell screening technique after three cycles of the "adsorption-elution-amplification"procedure. Positive phage clones were identified by ELISA, and the inserted amino acid sequences in the displaying peptides were deduced from confirmation with DNA sequencing. The adhesion rate of ECs to monocytes was evaluated by cell counting. The activity of endothelial nitric oxide synthase (eNOS), and the expression levels of caveolin- 1 and intercellular adhesion molecule- 1 (ICAM- 1) were determined by Western blotting. Six positive clones specifically binding to injured ECV304 endothelial cells were selected from fourteen clones. Interestingly, four phages had peptides with tandem leucine, and two of these even shared an identical sequence. Functional analysis demonstrated that the YCPRYVRRKLENELLVL peptide shared by two clones inhibited the expression of ICAM-1, increased nitric oxide concentration in the culture media, and upregulated the expression of caveolin-1 and eNOS. As a result, the adhesion rate of monocytes to ECV304 cells was significantly reduced by 12.1%. These data suggest that the anti-adhesion effect of these novel peptides is related to the regulation of the caveolin-1/nitric oxide signal transduction pathway, and could be of use in potential therapeutic agents against certain cardiovascular diseases initiated by vascular endothelial cell damage.

  17. Uses of Phage Display in Agriculture: A Review of Food-Related Protein-Protein Interactions Discovered by Biopanning over Diverse Baits

    Directory of Open Access Journals (Sweden)

    Rekha Kushwaha

    2013-01-01

    Full Text Available This review highlights discoveries made using phage display that impact the use of agricultural products. The contribution phage display made to our fundamental understanding of how various protective molecules serve to safeguard plants and seeds from herbivores and microbes is discussed. The utility of phage display for directed evolution of enzymes with enhanced capacities to degrade the complex polymers of the cell wall into molecules useful for biofuel production is surveyed. Food allergies are often directed against components of seeds; this review emphasizes how phage display has been employed to determine the seed component(s contributing most to the allergenic reaction and how it has played a central role in novel approaches to mitigate patient response. Finally, an overview of the use of phage display in identifying the mature seed proteome protection and repair mechanisms is provided. The identification of specific classes of proteins preferentially bound by such protection and repair proteins leads to hypotheses concerning the importance of safeguarding the translational apparatus from damage during seed quiescence and environmental perturbations during germination. These examples, it is hoped, will spur the use of phage display in future plant science examining protein-ligand interactions.

  18. Uses of phage display in agriculture: a review of food-related protein-protein interactions discovered by biopanning over diverse baits.

    Science.gov (United States)

    Kushwaha, Rekha; Payne, Christina M; Downie, A Bruce

    2013-01-01

    This review highlights discoveries made using phage display that impact the use of agricultural products. The contribution phage display made to our fundamental understanding of how various protective molecules serve to safeguard plants and seeds from herbivores and microbes is discussed. The utility of phage display for directed evolution of enzymes with enhanced capacities to degrade the complex polymers of the cell wall into molecules useful for biofuel production is surveyed. Food allergies are often directed against components of seeds; this review emphasizes how phage display has been employed to determine the seed component(s) contributing most to the allergenic reaction and how it has played a central role in novel approaches to mitigate patient response. Finally, an overview of the use of phage display in identifying the mature seed proteome protection and repair mechanisms is provided. The identification of specific classes of proteins preferentially bound by such protection and repair proteins leads to hypotheses concerning the importance of safeguarding the translational apparatus from damage during seed quiescence and environmental perturbations during germination. These examples, it is hoped, will spur the use of phage display in future plant science examining protein-ligand interactions.

  19. A mimotope of Pre-S2 region of surface antigen of viral hepatitis Bscreened by phage display

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    To acquire the phage-displayed mimotopes which mimic the specificity of hepatitis B virus surface antigen (HBsAg), a random peptide library expressing linear peptide with 12 amino acids in length were used to screen with the serum from a hepatitis B virus infected patient in the recovery phase. After 3 rounds of biopanning, the positive phages were confirmed by competitive ELISA using HBsAg/P33. Two phagotopes were identified and one of them was confirmed as mimotope by competition experiment. Based on the mimotpe, a multiple antigenic peptide with four branches was synthesized by solid phase peptide synthesis. The antiginicity and specificity of the synthesized antigen was tested in BALB/c mice compared with the native epitope-based antigen. The results showed that the mimotope-based antigen could evoke higher titer of antibodies with the same specificity of the epitope-based antigen. Those findings indicate mimotopes can be used in antigen and vaccine design.

  20. Antagonist peptides of human interferon-α2b isolated from phage display library inhibit interferon induced antiviral activity

    Institute of Scientific and Technical Information of China (English)

    Wang TIAN; Gang BAI; Zheng-he LI; Wen-bo YANG

    2006-01-01

    Aim: To screen human interferon (IFN)-α2b antagonist peptides from a phage displayed heptapeptide library. Methods: WISH cells and polyclonal anti-IFN-α2b antibodies were used to select IFN receptor-binding peptides from a phage displayed heptapeptide library. The specific binding of phage clones was examined by phage ELISA and immunohistochemistry. The specific binding activities of synthetic peptides to WISH cells were detected by competition assay. Effects of synthetic peptides to IFN-induced antiviral activity were analyzed by evaluating the cytopathic effect (CPE) using the MTT method. Results: Twenty-three positive clones were obtained after seven rounds of selection. Ten clones were randomly picked from the positive clones and were sequenced. The corresponding amino acid sequences suggested 3 groups homologous to the 3 domains of IFN-α2b, defined by residues 24-41, 43-49, and 148-158 of IFN-α2b. As they presented as corresponding to IFN receptor-binding domains, AB loop and E helix, clone № 26 and 35 were chosen for further characterization and shown to bind to WISH cells. Two peptides corresponding to clone № 26 and 35, designated SP-7(SLSPGLP) and FY-7(FSAPVRY) were shown to compete with GFP-IFN-α2b for binding to its receptor and to inhibit the IFN-α2b-induced antiviral activity. Conclusion: Both IFN-α2b antagonist peptides, SP-7 and FY-7, were able to inhibit the IFN-induced antiviral activity, and could be helpful in laying the foundation for the molecular mechanism of the interaction between IFN and its receptor.

  1. Bioadsorption strategies with yeast molecular display technology.

    Science.gov (United States)

    Shibasaki, Seiji; Ueda, Mitsuyoshi

    2014-01-01

    Molecular display techniques using microbial cell surfaces have been widely developed in the past twenty years, and are useful tools as whole cell catalysts for various applications such as bioconversion, bioremediation, biosensing, and the screening system of protein libraries. Furthermore, different types of microbial cells among eukaryotic and prokaryotic strains have been investigated for their use in surface display technologies. Recently, several kinds of protein-displaying yeasts have been utilized as bioadsorbents in this platform technology. In particular, these trials have successfully expanded the possibility of applications to metal binding, affinity purification, and receptor-ligand interaction by using the yeast cell surface. In this mini review, we describe the general principles of molecular display technology using yeast cells and its applications, with a particular focus on bioadsorption.

  2. Monoclonal antibody proteomics: use of antibody mimotope displaying phages and the relevant synthetic peptides for mAb scouting.

    Science.gov (United States)

    Hajdú, István; Flachner, Beáta; Bognár, Melinda; Végh, Barbara M; Dobi, Krisztina; Lőrincz, Zsolt; Lázár, József; Cseh, Sándor; Takács, László; Kurucz, István

    2014-08-01

    Monoclonal antibody proteomics uses nascent libraries or cloned (Plasmascan™, QuantiPlasma™) libraries of mAbs that react with individual epitopes of proteins in the human plasma. At the initial phase of library creation, cognate protein antigen and the epitope interacting with the antibodies are not known. Scouting for monoclonal antibodies (mAbs) with the best binding characteristics is of high importance for mAb based biomarker assay development. However, in the absence of the identity of the cognate antigen the task represents a challenge. We combined phage display, and surface plasmon resonance (Biacore) experiments to test whether specific phages and the respective mimotope peptides obtained from large scale studies are applicable to determine key features of antibodies for scouting. We show here that mAb captured phage-mimotope heterogeneity that is the diversity of the selected peptide sequences, is inversely correlated with an important binding descriptor; the off-rate of the antibodies and that represents clues for driving the selection of useful mAbs for biomarker assay development. Carefully chosen synthetic mimotope peptides are suitable for specificity testing in competitive assays using the target proteome, in our case the human plasma.

  3. Advanced manufacturing technologies on color plasma displays

    Science.gov (United States)

    Betsui, Keiichi

    2000-06-01

    The mass production of the color plasma display started from 1996. However, since the price of the panel is still expensive, PDPs are not in widespread use at home. It is necessary to develop the new and low-cost manufacturing technologies to reduce the price of the panel. This paper describes some of the features of new fabrication technologies of PDPs.

  4. Affinity isolation of antigen-specific circulating B cells for generation of phage display-derived human monoclonal antibodies

    DEFF Research Database (Denmark)

    Ditzel, Henrik

    2009-01-01

    A method is described for affinity isolation of antigen-specific circulating B cells of interest for subsequent generation of immune antibody phage display libraries. This approach should overcome the problem of low yields of monoclonal antibodies of interest in the libraries generated from...... peripheral blood lymphocytes caused by the low abundance of antigen-specific B cells in the circulation. The preselection of B cells is based on the specificity of the surface Ig receptor and is accomplished using the antigen of interest conjugated to magnetic beads. This method should significantly increase...

  5. SINGLE CHAIN VARIABLE FRAGMENTS OF ANTIBODIES AGAINST DIPHTHERIA TOXIN B-SUBUNIT ISOLATED FROM PHAGE DISPLAY HUMAN ANTIBODY LIBRARY

    OpenAIRE

    Oliinyk O. S.; Kaberniuk A. A.; Kolibo D. V.; Komisarenko S. V.

    2014-01-01

    Diphtheria toxin is an exoantigen of Corynebacterium diphtheriae that inhibits protein synthesis and kills sensitive cells. The aim of this study was to obtain human recombinant single-chain variable fragment (scFv) antibodies against receptor-binding B subunit of diphtheria toxin. 12 specific clones were selected after three rounds of a phage display naїve (unimmunized) human antibody library against recombinant B-subunit. scFv DNA inserts from these 12 clones were digested with MvaI, an...

  6. Identification of Hitherto Undefined B-Cell Epitopes by Antibodies in the Sera of Vitiligo Patients Using Phage-Display Peptide Library

    Directory of Open Access Journals (Sweden)

    Zohreh Jadali

    2003-12-01

    Full Text Available A random 12 mers phage library was used to screen a pool of immunoglo¬bulin fractions obtained from vitiligo patients. Subsequent to panning experiments, a panel of affinity selected phage from vitiligo patients were obtained. This panel was tested using an ELIS A for their reactivity with pooled sera from patients and normal controls. Among the 16 randomly selected clones, two of clones showed distinct positive reactivity with the patient's sera compared with controls. The peptides displayed by these phages expressed the following amino acid sequences: SHMPLANQYQWA and NHVQAWEQFWDS. Thus, screening with phage-displayed random peptide library of vitiligo sera can reveal peptide sequences that mimic vitiligo-related self-antigen.

  7. Routing and advanced display technologies within STOMPM

    Science.gov (United States)

    Mittu, Ranjeev; Uhlmann, Jeffrey K.; McCune, Justin

    1998-08-01

    This paper will discuss research conducted at the Naval Research Laboratory in the area of automated routing, advanced 3D displays and novel interface techniques for interacting with those displays. This research has culminated in the development of the strike optimized mission planing module (STOMPM). The STOMPM testbed incorporates new technologies/results in the aforementioned areas to address the deficiencies in current systems and advance the state of the art in military planing systems.

  8. A large semi-synthetic single-chain Fv phage display library based on chicken immunoglobulin genes

    Directory of Open Access Journals (Sweden)

    Jordaan Frances

    2004-04-01

    Full Text Available Abstract Background Antibody fragments selected from large combinatorial libraries have numerous applications in diagnosis and therapy. Most existing antibody repertoires are derived from human immunoglobulin genes. Genes from other species can, however, also be used. Because of the way in which gene conversion introduces diversity, the naïve antibody repertoire of the chicken can easily be accessed using only two sets of primers. Results With in vitro diagnostic applications in mind, we have constructed a large library of recombinant filamentous bacteriophages displaying single chain antibody fragments derived from combinatorial pairings of chicken variable heavy and light chains. Synthetically randomised complementarity determining regions are included in some of the heavy chains. Single chain antibody fragments that recognise haptens, proteins and virus particles were selected from this repertoire. Affinities of three different antibody fragments were determined using surface plasmon resonance. Two were in the low nanomolar and one in the subnanomolar range. To illustrate the practical value of antibodies from the library, phage displayed single chain fragments were incorporated into ELISAs aimed at detecting African horsesickness and bluetongue virus particles. Virus antibodies were detected in a competitive ELISA. Conclusion The chicken-derived phage library described here is expected to be a versatile source of recombinant antibody fragments directed against a wide variety of antigens. It has the potential to provide monoclonal reagents with applications in research and diagnostics. For in vitro applications, naïve phage libraries based on avian donors may prove to be useful adjuncts to the selectable antibody repertoires that already exist.

  9. Using phage and yeast display to select hundreds of monoclonal antibodies: application to antigen 85, a tuberculosis biomarker.

    Directory of Open Access Journals (Sweden)

    Fortunato Ferrara

    Full Text Available BACKGROUND: Current diagnostic methods for tuberculosis (TB, a major global health challenge that kills nearly two million people annually, are time-consuming and inadequate. During infection a number of bacterial molecules that play a role in the infective process are released and have been proposed as biomarkers for early TB diagnosis. Antigen 85 (Ag85 is the most abundant secreted TB protein, and a potential target for this diagnostic approach. One of the bottlenecks in the direct detection of such bacterial targets is the availability of robust, sensitive, specific antibodies. METHODS: Using Ag85 as a model, we describe a method to select antibodies against any potential target using a novel combination of phage and yeast display that exploits the advantage of each approach. RESULTS: The efficiency of this approach was attested to by the 111 specific antibodies identified in initial screens. These were assessed for binding to the different Ag85 subunits, affinity, and activity in sandwich assays. CONCLUSIONS: The novelty of this approach lies in the possibility of screening the entire output of a phage antibody selection in a single experiment by yeast display. This can be considered analogous to carrying out a million ELISAs. The monoclonal antibodies (mAbs identified in this way show high binding affinity and selectivity for the antigens and offer an advantage over traditional mAbs produced by relatively expensive and time consuming techniques. This approach has wide applicability, and the affinity of selected antibodies can be significantly improved, if required.

  10. Generation of Potent Anti-Vascular Endothelial Growth Factor Neutralizing Antibodies from Mouse Phage Display Library for Cancer Therapy

    Directory of Open Access Journals (Sweden)

    Yan-Da Lai

    2016-02-01

    Full Text Available Vascular endothelial growth factor (VEGF is an important stimulator for angiogenesis in solid tumors. Blocking VEGF activity is an effective therapeutic strategy to inhibit tumor growth and metastasis. Avastin, a humanized monoclonal antibody recognizes VEGF, has been approved by the US Food and Drug Administration. To generate potential VEGF-recognizing antibodies with better tumor regression ability than that of Avastin, we have designed a systematic antibody selection plan. From mice immunized with recombinant human VEGF, we generated three phage display libraries, scFv-M13KO7, Fab-M13KO7, and scFv-Hyperphage, in single-chain Fv (scFv or Fab format, displayed using either M13KO7 helper phage or Hyperphage. Solid-phase and solution-phase selection strategies were then applied to each library, generating six panning combinations. A total of sixty-four antibodies recognizing VEGF were obtained. Based on the results of epitope mapping, binding affinity, and biological functions in tumor inhibition, eight antibodies were chosen to examine their abilities in tumor regression in a mouse xenograft model using human COLO 205 cancer cells. Three of them showed improvement in the inhibition of tumor growth (328%–347% tumor growth ratio (% of Day 0 tumor volume on Day 21 vs. 435% with Avastin. This finding suggests a potential use of these three antibodies for VEGF-targeted therapy.

  11. Specific probe selection from landscape phage display library and its application in enzyme-linked immunosorbent assay of free prostate-specific antigen.

    Science.gov (United States)

    Lang, Qiaolin; Wang, Fei; Yin, Long; Liu, Mingjun; Petrenko, Valery A; Liu, Aihua

    2014-03-01

    Probes against targets can be selected from the landscape phage library f8/8, displaying random octapeptides on the pVIII coat protein of the phage fd-tet and demonstrating many excellent features including multivalency, stability, and high structural homogeneity. Prostate-specific antigen (PSA) is usually determined by immunoassay, by which antibodies are frequently used as the specific probes. Herein we found that more advanced probes against free prostate-specific antigen (f-PSA) can be screened from the landscape phage library. Four phage monoclones were selected and identified by the specificity array. One phage clone displaying the fusion peptide ERNSVSPS showed good specificity and affinity to f-PSA and was used as a PSA capture probe in a sandwich enzyme-linked immunosorbent assay (ELISA) array. An anti-human PSA monoclonal antibody (anti-PSA mAb) was used to recognize the captured antigen, followed by horseradish peroxidase-conjugated antibody (HRP-IgG) and o-phenylenediamine, which were successively added to develop plate color. The ELISA conditions such as effect of blocking agent, coating buffer pH, phage concentration, antigen incubation time, and anti-PSA mAb dilution for phage ELISA were optimized. On the basis of the optimal phage ELISA conditions, the absorbance taken at 492 nm on a microplate reader was linear with f-PSA concentration within 0.825-165 ng/mL with a low limit of detection of 0.16 ng/mL. Thus, the landscape phage is an attractive biomolecular probe in bioanalysis.

  12. Human antibody fragments specific for the epidermal growth factor receptor selected from large non-immunised phage display libraries.

    Science.gov (United States)

    Souriau, Christelle; Rothacker, Julie; Hoogenboom, Hennie R; Nice, Edouard

    2004-09-01

    Antibodies to EGFR have been shown to display anti-tumour effects mediated in part by inhibition of cellular proliferation and angiogenesis, and by enhancement of apoptosis. Humanised antibodies are preferred for clinical use to reduce complications with HAMA and HAHA responses frequently seen with murine and chimaeric antibodies. We have used depletion and subtractive selection strategies on cells expressing the EGFR to sample two large antibody fragment phage display libraries for the presence of human antibodies which are specific for the EGFR. Four Fab fragments and six scFv fragments were identified, with affinities of up to 2.2nM as determined by BIAcore analysis using global fitting of the binding curves to obtain the individual rate constants (ka and kd). This overall approach offers a generic screening method for the identification of growth factor specific antibodies and antibody fragments from large expression libraries and has potential for the rapid development of new therapeutic and diagnostic reagents.

  13. Utilization of Multi-Immunization and Multiple Selection Strategies for Isolation of Hapten-Specific Antibodies from Recombinant Antibody Phage Display Libraries

    Directory of Open Access Journals (Sweden)

    Antti Tullila

    2017-05-01

    Full Text Available Phage display technology provides a powerful tool for the development of novel recombinant antibodies. In this work, we optimized and streamlined the recombinant antibody discovery process for haptens as an example. A multi-immunization approach was used in order to avoid the need for construction of multiple antibody libraries. Selection methods were developed to utilize the full potential of the recombinant antibody library by applying four different elution conditions simultaneously. High-throughput immunoassays were used to analyse the binding properties of the individual antibody clones. Different carrier proteins were used in the immunization, selection, and screening phases to avoid enrichment of the antibodies for the carrier protein epitopes. Novel recombinant antibodies against mycophenolic acid and ochratoxin A, with affinities up to 39 nM and 34 nM, respectively, were isolated from a multi-immunized fragment antigen-binding (Fab library.

  14. Utilization of Multi-Immunization and Multiple Selection Strategies for Isolation of Hapten-Specific Antibodies from Recombinant Antibody Phage Display Libraries

    Science.gov (United States)

    Tullila, Antti; Nevanen, Tarja K.

    2017-01-01

    Phage display technology provides a powerful tool for the development of novel recombinant antibodies. In this work, we optimized and streamlined the recombinant antibody discovery process for haptens as an example. A multi-immunization approach was used in order to avoid the need for construction of multiple antibody libraries. Selection methods were developed to utilize the full potential of the recombinant antibody library by applying four different elution conditions simultaneously. High-throughput immunoassays were used to analyse the binding properties of the individual antibody clones. Different carrier proteins were used in the immunization, selection, and screening phases to avoid enrichment of the antibodies for the carrier protein epitopes. Novel recombinant antibodies against mycophenolic acid and ochratoxin A, with affinities up to 39 nM and 34 nM, respectively, were isolated from a multi-immunized fragment antigen-binding (Fab) library. PMID:28561803

  15. Interactive displays natural human-interface technologies

    CERN Document Server

    Bhowmik, Achintya K

    2014-01-01

    One of the first books to provide an in-depth discussion of the technologies, applications and trends in the rapidly emerging field of interactive displays (touch, gesture & voice) The book will cover the technologies, applications and trends in the field of interactive displays, namely interfaces based on touch, gesture and voice and those using a combination of these technologies. The book will be split into 4 main parts with each being dedicated to a specific user interface. Part 1 ''Touch Interfaces'' will provide a review of the currently deployed touch-screen technologies and applications. It will also cover the recent developments towards achieving thinner, lightweight and cost-reduced touch screen panels in the future via integration of touch functionalities. Part 2 ''Gesture Interfaces'' will examine techniques and applications in stereoscopic 3D computer vision, structured-light 3D computer vision and time-of-flight 3D computer vision in gesture interfaces. Part 3 ''Voice Interfaces'' will revie...

  16. Mobile display technologies: Past developments, present technologies, and future opportunities

    Science.gov (United States)

    Ohshima, Hiroyuki

    2014-01-01

    It has been thirty years since the first active matrix (AM) flat panel display (FPD) was industrialized for portable televisions (TVs) in 1984. The AM FPD has become a dominant electronic display technology widely used from mobile displays to large TVs. The development of AM FPDs for mobile displays has significantly changed our lives by enabling new applications, such as notebook personal computers (PCs), smartphones and tablet PCs. In the future, the role of mobile displays will become even more important, since mobile displays are the live interface for the world of mobile communications in the era of ubiquitous networks. Various developments are being conducted to improve visual performance, reduce power consumption and add new functionality. At the same time, innovative display concepts and novel manufacturing technologies are being investigated to create new values.

  17. Electronic control/display interface technology

    Science.gov (United States)

    Parrish, R. V.; Busquets, A. M.; Murray, R. F.; Hatfield, J. J.

    1985-01-01

    An effort to produce a representative workstation for the Space Station Data Management Test Bed that provides man/machine interface design options for consolidating, automating, and integrating the space station work station, and hardware/software technology demonstrations of space station applications is discussed. The workstation will emphasize the technologies of advanced graphics engines, advanced display/control medias, image management techniques, multifunction controls, and video disk utilizations.

  18. Isolation and Evaluation of Specific Human Recombinant Antibodies from a Phage Display Library against HER3 Cancer Signaling Antigen

    Directory of Open Access Journals (Sweden)

    Foroogh Nejatollahi

    2014-07-01

    Full Text Available Background: The human epidermal growth factor receptor family comprises four homologous members: EGFR (ErbB1, ErbB2 (HER2, ErbB3 (HER3 and ErbB4 (HER4.This family plays an important role in the signaling pathway and cell proliferation. The heterodimerization of HER2 with HER3 leads to tumor cell proliferation. Monoclonal antibody to the human HER3 receptor blocks HER3 heterodimerization and inhibits the growth of breast cancer cells. Due to their human origin, small size, rapid penetration and high affinity properties, recombinant single chain antibodies (scFv have been introduced as the most desired agents for cancer immunotherapy. In this study, we use a phage display system to select specific scFvs against HER3 for their use in cancer targeted therapy. Methods: A phage antibody display library of scFv was panned against an immunodominant epitope of HER3. Phage rescue was performed on the library. The supernatant that contained the appropriate scFv (109 PFU/ml was added to an immunotube which was coated with the peptide. Elution was done using log phase E. coli TG1. The clones were amplified by PCR and DNA fingerprinted to select the specific clones against the epitope. The specificity of the selected antibodies was tested in ELISA. Results: The results represented two predominant patterns with the frequency of 25%. The other patterns showed the frequencies of 5%-10%. scFv1 and scFv2 demonstrated positive ELISA with absorbances of 0.63 and 0.46, respectively while the absorbances of wells without peptide were 0.19 and 0.11, respectively. Conclusion: In this study two specific scFvs were selected against HER3 antigen in a successful panning process. Phage ELISA represented the specific binding of scFvs against HER3.The selected scFvs reacted only with the corresponding peptides. However, no reaction with the other peptides was detected. The selected anti-HER3 scFvs have suggested that these human high affinity and small antibodies that bind

  19. Targeting Leishmania major parasite with peptides derived from a combinatorial phage display library.

    Science.gov (United States)

    Rhaiem, Rafik Ben; Houimel, Mehdi

    2016-07-01

    Cutaneous leishmaniasis (CL) is a global problem caused by intracellular protozoan pathogens of the genus Leishmania for which there are no suitable vaccine or chemotherapy options. Thus, de novo identification of small molecules binding to the Leishmania parasites by direct screening is a promising and appropriate alternative strategy for the development of new drugs. In this study, we used a random linear hexapeptide library fused to the gene III protein of M13 filamentous bacteriophage to select binding peptides to metacyclic promastigotes from a highly virulent strain of Leishmania major (Zymodeme MON-25; MHOM/TN/94/GLC94). After four rounds of stringent selection and amplification, polyclonal and monoclonal phage-peptides directed against L. major metacyclic promastigotes were assessed by ELISA, and the optimal phage-peptides were grown individually and characterized for binding to L. major by monoclonal phage ELISA. The DNA of 42 phage-peptides clones was amplified by PCR, sequenced, and their amino acid sequences deduced. Six different peptide sequences were obtained with frequencies of occurrence ranging from 2.3% to 85.7%. The biological effect of the peptides was assessed in vitro on human monocytes infected with L. major metacyclic promastigotes, and in vivo on susceptible parasite-infected BALB/c mice. The development of cutaneous lesions in the right hind footpads of infected mice after 13 weeks post-infection showed a protection rate of 81.94% with the injected peptide P2. Moreover, Western blots revealed that the P2 peptide interacted with the major surface protease gp63, a protein of 63kDa molecular weight. Moreover, bioinformatics were used to predict the interaction between peptides and the major surface molecule of the L. major. The molecular docking showed that the P2 peptide has the minimum interaction energy and maximum shape complimentarity with the L. major gp63 active site. Our study demonstrated that the P2 peptide occurs at high frequency

  20. Re-engineering of the PAM1 phage display monoclonal antibody to produce a soluble, versatile anti-homogalacturonan scFv

    DEFF Research Database (Denmark)

    Manfield, I. W.; Bernal Giraldo, Adriana Jimena; Møller, I.

    2006-01-01

    Antibody phage display is an increasingly important alternative method for the production of monoclonal antibodies (mAbs) and involves the expression of antibody fragments (scFvs) at the surface of bacteriophage particles. We have previously used this technique to generate a phage mAb (PAM1phage...... of the PAM1 mAb, we describe here the production of a phage-free, soluble scFv version of the PAM1 mAb (PAM1scFv). Using the new PAM1scFv probe, the occurrence of the HG epitope recognized can now be localized with high resolution within micro-domains of plant cell walls....

  1. Dissecting the Binding Mode of Low Affinity Phage Display Peptide Ligands to Protein Targets by Hydrogen/Deuterium Exchange Coupled to Mass Spectrometry

    DEFF Research Database (Denmark)

    Leurs, Ulrike; Lohse, Brian; Ming, Shonoi A;

    2014-01-01

    Phage display (PD) is frequently used to discover peptides capable of binding to biological protein targets. The structural characterization of peptide-protein complexes is often challenging due to their low binding affinities and high structural flexibility. Here, we investigate the use of hydro......Phage display (PD) is frequently used to discover peptides capable of binding to biological protein targets. The structural characterization of peptide-protein complexes is often challenging due to their low binding affinities and high structural flexibility. Here, we investigate the use...

  2. Generation and validation of a Shewanella oneidensis MR-1 clone set for protein expression and phage display.

    Directory of Open Access Journals (Sweden)

    Haichun Gao

    Full Text Available A comprehensive gene collection for S. oneidensis was constructed using the lambda recombinase (Gateway cloning system. A total of 3584 individual ORFs (85% have been successfully cloned into the entry plasmids. To validate the use of the clone set, three sets of ORFs were examined within three different destination vectors constructed in this study. Success rates for heterologous protein expression of S. oneidensis His- or His/GST-tagged proteins in E. coli were approximately 70%. The ArcA and NarP transcription factor proteins were tested in an in vitro binding assay to demonstrate that functional proteins can be successfully produced using the clone set. Further functional validation of the clone set was obtained from phage display experiments in which a phage encoding thioredoxin was successfully isolated from a pool of 80 different clones after three rounds of biopanning using immobilized anti-thioredoxin antibody as a target. This clone set complements existing genomic (e.g., whole-genome microarray and other proteomic tools (e.g., mass spectrometry-based proteomic analysis, and facilitates a wide variety of integrated studies, including protein expression, purification, and functional analyses of proteins both in vivo and in vitro.

  3. Identification of a novel peptide ligand targeting visceral adipose tissue via transdermal route by in vivo phage display.

    Science.gov (United States)

    Lee, Nam Kyung; Kim, Hong Shin; Kim, Kyung Hyun; Kim, Eun-Bae; Cho, Chong Su; Kang, Sang Kee; Choi, Yun Jaie

    2011-11-01

    To find novel peptide ligands targeting visceral adipose tissue (visceral fat) via transdermal route, in vivo phage display screening was conducted by dermal administration of a phage-peptide library to rats and a peptide sequence, CGLHPAFQC (designated as TDA1), was identified as a targeting ligand to visceral adipose tissue through the consecutive transdermal biopannings. Adipocyte-specific affinity and transdermal activity of the TDA1 were validated in vitro and targeting ability of the dermally administered TDA1 to visceral adipose tissue was also confirmed in vivo. TDA1 was effectively translocated into systemic circulation after dermal administration and selectively targeted visceral adipose tissue without any preference to other organs tested. Fluorescent microscopic analysis revealed that the TDA1 could be specifically localized in the hair follicles of the skin, as well as in the visceral adipose tissue. Thus, we inferred that dermally administered TDA1 would first access systemic circulation via hair follicles as its transdermal route and then could target visceral fat effectively. The overall results suggest that the TDA1 peptide could be potentially applied as a homing moiety for delivery of anti-obesity therapeutics to visceral fat through the convenient transdermal pathway.

  4. Targeting essential Eimeria ninakohlyakimovae sporozoite ligands for caprine host endothelial cell invasion with a phage display peptide library.

    Science.gov (United States)

    Ruiz, A; Pérez, D; Muñoz, M C; Molina, J M; Taubert, A; Jacobs-Lorena, M; Vega-Rodríguez, J; López, A M; Hermosilla, C

    2015-11-01

    Eimeria ninakohlyakimovae is an important coccidian parasite of goats which causes severe diarrhoea in young animals. Specific molecules that mediate E. ninakohlyakimovae host interactions and molecular mechanisms involved in the pathogenesis are still unknown. Although strong circumstantial evidence indicates that E. ninakohlyakimovae sporozoite interactions with caprine endothelial host cells (ECs) are specific, hardly any information is available about the interacting molecules that confer host cell specificity. In this study, we describe a novel method to identify surface proteins of caprine umbilical vein endothelial cells (CUVEC) using a phage display library. After several panning rounds, we identified a number of peptides that specifically bind to the surface of CUVEC. Importantly, caprine endothelial cell peptide 2 (PCEC2) and PCEC5 selectively reduced the infection rate by E. ninakohlyakimovae sporozoites. These preliminary data give new insight for the molecular identification of ligands involved in the interaction between E. ninakohlyakimovae sporozoites and host ECs. Further studies using this phage approach might be useful to identify new potential target molecules for the development of anti-coccidial drugs or even new vaccine strategies.

  5. Phage display selection on whole cells yields a small peptide specific for HCV receptor human CD81

    Institute of Scientific and Technical Information of China (English)

    JIE CAO; PING ZHAO; X1AO HUI MIAO; LAN JUAN ZHAO; LI JUN XUE; ZHONG TIAN QI

    2003-01-01

    The human CD81(hCD81),the most recently proposed receptor of hepatitis C virus(HCV),can especifically bind to HCV envelope glycoprotein 2(E2).In this study,hCD81-expressing murine NIH/3T3 cells were used to select hCD81-binding peptides from a phage displayed nonapeptide library(PVIII9aaCys).Eighteen of the 75clones selected from the library showed specific binding to the hCD81-expressing NIH/3T3 cells by enzyme linked immunosorbent assay(ELISA)and competitive inhibition test.Twelve out of the 18 clones shared the amino acid motif SPQYWTGPA.Sequence comparison of the motif showed no amino acid homology with the native HCV E2.The motif-containing phages could competitively inhibit the ability of HCV E2 binding to native hCD81-expressing MOLT-4 cells,and induce HCV E2 specific immune response in vivo.These results suggest that the selected motif SPQYWTGPA should be a mimotope of HCV E2 to bind to hCD81 molecules.Our findings cast new light on developing HCV receptor antagonists.

  6. Identification of a cardiac specific protein transduction domain by in vivo biopanning using a M13 phage peptide display library in mice.

    Directory of Open Access Journals (Sweden)

    Maliha Zahid

    Full Text Available BACKGROUND: A peptide able to transduce cardiac tissue specifically, delivering cargoes to the heart, would be of significant therapeutic potential for delivery of small molecules, proteins and nucleic acids. In order to identify peptide(s able to transduce heart tissue, biopanning was performed in cell culture and in vivo with a M13 phage peptide display library. METHODS AND RESULTS: A cardiomyoblast cell line, H9C2, was incubated with a M13 phage 12 amino acid peptide display library. Internalized phage was recovered, amplified and then subjected to a total of three rounds of in vivo biopanning where infectious phage was isolated from cardiac tissue following intravenous injection. After the third round, 60% of sequenced plaques carried the peptide sequence APWHLSSQYSRT, termed cardiac targeting peptide (CTP. We demonstrate that CTP was able to transduce cardiomyocytes functionally in culture in a concentration and cell-type dependent manner. Mice injected with CTP showed significant transduction of heart tissue with minimal uptake by lung and kidney capillaries, and no uptake in liver, skeletal muscle, spleen or brain. The level of heart transduction by CTP also was greater than with a cationic transduction domain. CONCLUSIONS: Biopanning using a peptide phage display library identified a peptide able to transduce heart tissue in vivo efficiently and specifically. CTP could be used to deliver therapeutic peptides, proteins and nucleic acid specifically to the heart.

  7. PRODUCTION OF PHAGE-DISPLAYED ANTI-IDIOTYPIC ANTIBODY SINGLE CHAIN VARIABLE FRAGMENTS TO MG7 MONOCLONAL ANTIBODY DIRECTED AGAINST GASTRIC CARCINOMA

    Institute of Scientific and Technical Information of China (English)

    何凤田; 聂勇战; 陈宝军; 乔太东; 韩者艺; 樊代明

    2002-01-01

    Objective. To generate phage-displayed anti-idiotypic antibody single chain variable fragments (anti -Id ScFv) to MG7 monoclonal antibody (McAb) directed against gastric carcinoma so as to lay a foundation for developing anti-Id ScFv vaccine of the cancer.Methods. Balb/c mice were immunized i. p. with MG7 McAb conjugated with keyhole limpet hemocyanin (KLH), and mRNA was isolated from the spleens of the immunized mice. Heavy and light chain (VH and VL)genes of antibody were amplified separately and assembled into ScFv genes with a linker DNA by PCR. The ScFv genes were ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into competent E. coli TG1. The transformants were infected with M13KO7 helper phage to yield recombinant phages displaying ScFv on the tips of M13 phage. After 4 rounds of panning with MG7, the MG7-positive clones were selected by ELISA from the enriched phages. Thetypesoftheanti-IdScFvdisplayedontheselectedphagecloneswerepreliminarily identified by competition ELISA.Results. The VH, VL and ScFv DNAs were about 340 bp, 320 bp and 750 bp respectively. Twenty-four MG7-positive clones were selected from 60 enriched phage clones, among which 5 displayed β or γtype anti-Id ScFv.Conclsion. The anti-Id ScFv to MG7 McAb can be successfully selected by recombinant phage antibody technique, which paves a way for the study of prevention and cure of gastric carcinoma by using anti-Id ScFv.

  8. Identification of Target Ligands of CORYNE in Arabidopsis by Phage Display Library

    Institute of Scientific and Technical Information of China (English)

    Heng Zhao; Shuzhen Li; Jiping Sheng; Lin Shen; Yuhui Yang; Bin Yao

    2011-01-01

    CORYNE (CRN) plays important roles in stem cell division and differentiation of shoot apical meristem (SAM) in Arabidopsis thaliana. The cytoplasmic kinase domain of CRN has been cloned and expressed in Escherichia coil, and further purified by two consecutive steps of affinity chromatography. By using this purified CRN as a ligand, a 12-mer random-peptide library was used to determine the specific amino acid sequences binding with the recombinant CRN molecule. After four rounds of biopanning, positive phage clones were isolated and sequenced, and further tested by enzyme linked immunosorbent assay for their binding ability and specificity. Two positive clones that specifically bind to the intracellular protein kinase domain of CRN have been identified. Alignment of these peptides and the kinase-associated protein phosphatase (KAPP) shows high similarity, indicating that KAPP might interact with the cytoplasmic kinase domain of CRN and negatively regulate the CLV signal. Our current study would be helpful to better understand the CLV3 signal pathway.

  9. Analysis of pectic epitopes recognised by hybridoma and phage display monoclonal antibodies using defined oligosaccharides, polysaccharides, and enzymatic degradation

    DEFF Research Database (Denmark)

    Willats, William George Tycho; Limberg, G.; Buchholt, H.C.;

    2000-01-01

    The structure of epitopes recognised by anti-pectin monoclonal antibodies (mAbs) has been investigated using a series of model lime-pectin samples with defined degrees and patterns of methyl esterification, a range of defined oligogalacturonides and enzymatic degradation of pectic polysaccharides....... In immuno-dot-assays, the anti-homogalacturonan (HG) mAbs JIM5 and JIM7 both bound to samples with a wide range of degrees of methyl esterification in preference to fully de-esterified samples. In contrast, the anti-HG phage display mAb PAM1 bound most effectively to fully de-esterified pectin...... occurs where specific but undefined methyl-esterification patterns are present on HG domains, although fully de-esterified HG samples contain sub-optimal JIM5 epitopes. The persistence of mAb binding to epitopes in pectic antigens, with 41% blockwise esterification (P41) and 43% random esterification (F...

  10. Insight into screening immunoglobulin gene combinatorial libraries in a phage display vector: a tale of two antibodies.

    Science.gov (United States)

    Kakinuma, A; Portolano, S; Chazenbalk, G; Rapoport, B; McLachlan, S M

    1997-01-01

    Combinatorial libraries of immunoglobulin genes in "phage display" vectors are a powerful tool for obtaining antigen-specific antibody fragments. To date, this approach has been used to isolate abundant, but not rare, human autoantibodies of IgG class. We have compared the relative efficiencies of panning pComb3 libraries made from intrathyroidal plasma cells for abundant human autoantibodies to thyroid peroxidase (TPO) and rare autoantibodies to the thyrotropin receptor (TSHR). TPO-specific Fab were readily obtained from a library using three different forms of recombinant antigen, (i) purified TPO, (ii) impure TPO in culture medium and, (iii) TPO expressed on the surface of CHO cells. In contrast, TSHR-specific Fab were not isolated. This was the case despite repeated pannings of six libraries from three optimal patients (IgG/kappa and IgG/lambda libraries for each patient). Both purified recombinant TSHR and CHO cells expressing TSHR on their surface were used. Library enrichment was observed on some screenings. However, Fab expressed by individual clones or from enriched libraries were not specific as determined by (i) binding to purified, radio-labeled antigen, (ii) FACS analysis of TSHR on intact CHO cells and, (iii) inhibition of radiolabeled TSH binding. Remarkably, in screening for both TPO- and TSHR-specific Fab, neither library enrichment nor the retention of cDNA inserts of the correct size correlated with obtaining Fab with the antigenic specificity sought. Indeed, excellent enrichment could be observed with conditioned medium from untransfected cells. Our data suggest that the key to isolating rare antibodies from phage display libraries is not the creation of vast libraries of greater diversity or even the development of more stable vectors. Rather, success in this endeavor appears to require reducing the "noise" of non-specific clones in a moderately sized library.

  11. Application of phage display in selecting Tomato spotted wilt virus - specific single-chain antibodies (scFvs) for sensitive diagnosis in ELISA

    NARCIS (Netherlands)

    Griep, R.A.; Prins, M.; Twisk, van C.; Keller, H.J.H.G.; Kerschbaumer, R.J.; Kormelink, R.; Goldbach, R.W.; Schots, A.

    2000-01-01

    A panel of recombinant single-chain antibodies (scFvs) against structural proteins of Tomato spotted wilt virus (TSWV) was retrieved from a human combinatorial scFv antibody library using the novel phage display technique. After subcloning the encoding DNA sequences in the expression vector pSKAP/S,

  12. Mapping of epitopes for autoantibodies to the Type 1 diabetes autoantigen IA-2 by peptide phage display and molecular modelling: Overlap of antibody and T-cell determinants

    DEFF Research Database (Denmark)

    A. Dromey, James; Weenink, Sarah M.; Peters, Günther H.J.

    2004-01-01

    phage display and homology modeling to characterize the epitope of a monoclonal IA-2 Ab (96/3) from a human type 1 diabetic patient. This Ab competes for IA-2 binding with Abs from the majority of patients with type 1 diabetes and therefore binds a region close to common autoantibody epitopes. Alignment...

  13. An anti-tumor protein produced by Trichinella spiralis and identified by screening a T7 phage display library, induces apoptosis in human hepatoma H7402 cells

    Science.gov (United States)

    Trichinella spiralis infection confers effective resistance to tumor cell expansion. In this study, a T7 phage cDNA display library was constructed to express genes encoded by T. spiralis. Organic phase multi-cell screening was used to sort through candidate proteins in a transfected human chronic m...

  14. Application of phage display in selecting Tomato spotted wilt virus - specific single-chain antibodies (scFvs) for sensitive diagnosis in ELISA

    NARCIS (Netherlands)

    Griep, R.A.; Prins, M.; Twisk, van C.; Keller, H.J.H.G.; Kerschbaumer, R.J.; Kormelink, R.; Goldbach, R.W.; Schots, A.

    2000-01-01

    A panel of recombinant single-chain antibodies (scFvs) against structural proteins of Tomato spotted wilt virus (TSWV) was retrieved from a human combinatorial scFv antibody library using the novel phage display technique. After subcloning the encoding DNA sequences in the expression vector pSKAP/S,

  15. A novel method for in Situ detection of hydrolyzable casein fragments in a cheese matrix by antibody phage display technique and CLSM

    DEFF Research Database (Denmark)

    Duan, Zhi; Brüggemann, Dagmar Adeline; Siegumfeldt, Henrik

    2009-01-01

    A novel method to monitor in situ hydrolyzable casein fragments during cheese ripening by using immunofluorescent labeling and confocal laser scanning microscopy (CLSM) was developed. Monoclonal single chain variable fragments of antibody (scFvs) were generated by antibody phage display toward th...

  16. Peptide-binding motif prediction by using phage display library for SasaUBA*0301, a resistance haplotype of MHC class I molecule from Atlantic Salmon (Salmo salar)

    DEFF Research Database (Denmark)

    Zhao, Heng; Hermsen, Trudi; Stet, Rene J M;

    2008-01-01

    -terminus. Meanwhile, phage display peptide library encoding random 12mer peptides was also screened against beta(2)m/SasaUBA*0301. Eighty-five percentages of the corresponding peptides have an enrichment of leucine, methionine, valine, or isoleucine at the C-terminus. We predict that this particular allele of Salmon...

  17. Epitope mapping porcine reproductive and respiratory syndrome virus by phage display: the nsp2 fragment of the replicase polyprotein contains a cluster of B-cell epitopes

    DEFF Research Database (Denmark)

    Oleksiewicz, M.B.; Bøtner, Anette; Toft, P.;

    2001-01-01

    We screened phage display libraries of porcine reproductive and respiratory syndrome virus (PRRSV) protein fragments with sera from experimentally infected pigs to identify linear B-cell epitopes that are commonly recognized during infection in vivo. We identified 10 linear epitope sites (ES) 11...

  18. Schistosoma japonicum:Isolation and Identification of Peptides Mimicking Ferritin Epitopes from Phage Display Library

    Institute of Scientific and Technical Information of China (English)

    Lian-Fei TANG; Xin-Yuan YI; Xian-Fang ZENG; Lin-Qian WANG; Shun-Ke ZHANG

    2004-01-01

    In an attempt to isolate and identify the antigenic epitopes on ferritin of Schistosoma japonicum(SjFer)and to test their protective potentiality against Schistosomajaponicum(S.j),polyclonal antisera against SjFer was prepared to screen a 12-mer random peptide library.Three rounds of biopanning were performed and resulted in an enrichment.Six peptides selected randomly from the third elute were all found to be positive by evaluating the binding to anti-SjFer sera by ELISA and Western blotting.Three amino acid sequences were deduced from the six phage clones by sequencing.When they were used to vaccinate mice,the three peptides could induce significant reduction in adult worms(26.7% ,20.4%,and 25.9%)as well as in liver eggs per gram(LEPG)(40.0%,38.2%,and 40.8%).This result showed that three mimotopes on SjFer were obtained and they could induce significant protective efficacy against S.j.

  19. Selection of Peptide Inhibitor to Matrix Metalloproteinase-2 Using Phage Display and Its Effects on Pancreatic Cancer Cell lines PANC-1 and CFPAC-1

    Directory of Open Access Journals (Sweden)

    Gao Lu, Maqing Zheng, Yunxia Zhu, Min Sha, Yue Wu, Xiao Han

    2012-01-01

    Full Text Available Despite tremendous advances in cancer treatment and survival rates, pancreatic cancer remains one of the most deadly afflictions and the fourth leading cause of cancer deaths in the world. Matrix Metalloproteinases (MMPs are thought to be involved in cancer progression. Matrix metalloproteinase (MMP-2 is known to play a pivotal role in tumor invasion, metastasis and angiogenesis, and validated to be the anticancer target. Inhibition of MMP-2 activity is able to reduce the cancer cell invasion and suppress tumor growth in vivo. Two novel peptides, M204C4 and M205C4, which could specially inhibit MMP-2 activity, were identified by a phage display library screening. We showed that M204C4 and M205C4 inhibited the activity of MMP-2 in a dose dependent manner in vitro. Two peptides reduced MMP-2 mediated invasion of the pancreatic cancer cell lines PANC-1 and CFPAC-1, but not affected the expression and release of MMP-2. Furthermore, these two peptides could suppress tumor growth in vivo. Our results indicated that two peptides selected by phase display technology may be used as anticancer drugs in the future.

  20. Screening and identification of human ZnT8-specific single-chain variable fragment (scFv) from type 1 diabetes phage display library.

    Science.gov (United States)

    Wu, Qian; Wang, Xiaodong; Gu, Yong; Zhang, Xiao; Qin, Yao; Chen, Heng; Xu, Xinyu; Yang, Tao; Zhang, Mei

    2016-07-01

    Zinc transporter 8 (ZnT8) is a major autoantigen and a predictive marker in type 1 diabetes (T1D). To investigate ZnT8-specific antibodies, a phage display library from T1D was constructed and single-chain antibodies against ZnT8 were screened and identified. Human T1D single-chain variable fragment (scFv) phage display library consists of approximately 1×10(8) clones. After four rounds of bio-panning, seven unique clones were positive by phage ELISA. Among them, C27 and C22, which demonstrated the highest affinity to ZnT8, were expressed in Escherichia coli Top10F' and then purified by affinity chromatography. C27 and C22 specifically bound ZnT8 N/C fusion protein and ZnT8 C terminal dimer with one Arg325Trp mutation. The specificity to human islet cells of these scFvs were further confirmed by immunohistochemistry. In conclusion, we have successfully constructed a T1D phage display antibody library and identified two ZnT8-specific scFv clones, C27 and C22. These ZnT8-specific scFvs are potential agents in immunodiagnostic and immunotherapy of T1D.

  1. Identification of amino acids involved in the Flo11p-mediated adhesion of Saccharomyces cerevisiae to a polystyrene surface using phage display with competitive elution

    DEFF Research Database (Denmark)

    Mortensen, Henrik Dam; Dupont, Kitt; Jespersen, Lene;

    2007-01-01

    . cerevisiae FLO11 wild-type (TBR1) cells had a higher consensus than those from competitive panning with S. cerevisiae flo11¿ mutant (TBR5) cells, suggesting that the wild-type cells interact with the plastic surface in a stronger and more similar way than the mutant cells. Tryptophan and proline were more...... a phage with a hydrophobic peptide containing no tryptophan and only two proline residues. Conclusions: Our results suggest a key role of tryptophan and proline in the hydrophobic interactions between Flo11p on the S. cerevisiae cell surface and the PolySorp surface. Significance and Impact of the Study......Aims: To identify the main amino acids involved in the Flo11p-mediated adhesion of Saccharomyces cerevisiae to the polystyrene surface PolySorp. Methods and Results: Using a combination of phage display and competitive elution revealed that 12-mer peptides of phages from competitive panning with S...

  2. Construction of an artificially randomized IgNAR phage display library: screening of variable regions that bind to hen egg white lysozyme.

    Science.gov (United States)

    Ohtani, Maki; Hikima, Jun-ichi; Jung, Tae Sung; Kondo, Hidehiro; Hirono, Ikuo; Aoki, Takashi

    2013-02-01

    To develop a multi-antigen-specific immunoglobulin new antigen receptor (IgNAR) variable (V) region phage display library, CDR3 in the V region of IgNAR from banded houndshark (Triakis scyllium) was artificially randomized, and clones specific for hen egg white lysozyme (HEL) were obtained by the biopanning method. The nucleotide sequence of CDR3 in the V region was randomly rearranged by PCR. Randomized CDR3-containing segments of the V region were ligated into T7 phage vector to construct a phage display library and resulted in a phage titer of 3.7 × 10(7) PFU/ml. Forty clones that contained randomized CDR3 inserts were sequenced and shown to have different nucleotide sequences. The HEL-specific clones were screened by biopanning using HEL-coated ELISA plates. After six rounds of screening, nine clones were identified as HEL-specific, eight of which showed a strong affinity to HEL in ELISA compared to a negative control (i.e., empty phage clone). The deduced amino acid sequences of CDR3 from the HEL-specific phage clones fell into four types (I-IV): type I contains a single cysteine residue and type II-IV contain two cysteine residues. These results indicated that the artificially randomized IgNAR library is useful for the rapid isolation of antigen-specific IgNAR V region without immunization of target antigen and showed that it is possible to isolate an antigen-specific IgNAR V region from this library.

  3. Analysis of proteins that interact with nucleocapsid protein of SARS-CoV using 15-mer phage-displayed library

    Institute of Scientific and Technical Information of China (English)

    LIU ZhengXue; WANG ZhanHui; LIU YingLe; DONG Wei; QI YiPeng

    2007-01-01

    Analysis of proteins that interact with N protein of SARS-CoV using 15-mer phage-displayed library will help to explore the virus pathogenesis and to develop new drugs and vaccines against SARS. In this study, we cloned, expressed and purified N protein of SARS-CoV. This 46-kD N protein was verified by SDS-PAGE and Western-blot. Then, the peptides binding-specific to N protein were identified using 15-mer phage-displayed library. Surprisingly, all of the 89 clones from monoclonal ELISA were positive (S/N>2.1) and the result was further confirmed experimentally once again. Six N protein-binding peptides, designated separately as SNA1, SNA2, SNA4, SNA5, SNA9 and SNG11, were selected for sequencing. Sequence analysis suggested that SNA5 shared approximatively 100% sequence identity to SNA4, SNA2, SNA9 and SNA1. In addition, the binding specificity of the 15-mer peptides with the SARS-CoV N protein was further demonstrated by blocking ELISA using the synthetical 15-mer peptide according to the deduced amino acid sequence of SNA5. Also, the deduced amino sequence of SNA5 was compared with proteins in translated database using the tblastx program, and the results showed that the proteins with the highest homology were Ubiquinol-cytochrome c reductase iron-sulfur subunits (UCRI or UQCR), otherwise known as the Rieske iron-sulfur proteins (RISP). Notablely, in the [2Fe-2S] redox centre of UCRI, there were 6 residues [GGW(Y)F(Y)CP] compatible to the residues (position 2→7, GGWFCP7) of the NH2-terminal of the 15-mer peptide, which indicated higher binding specificity between the N protein of SARS-CoV and the redox centre of UCRI to some extent. Here, the possible molecular mechanisms of SARS-CoV N protein in the pathogenesis of SARS are discussed.

  4. Schistosoma japonicum:construction of phage display antibody library and its application in the immunodiagnosis of infection

    Institute of Scientific and Technical Information of China (English)

    陈代雄; 何蔼; 詹希美; 俞慕华; 雷智刚; 孟锦绣; 李卓雅; 梁瑜; 张瑞琳

    2004-01-01

    Background A monoclonal antibody would be an effective tool for the detection of circulating antigens in the serum of patients with schistosomiasis, but the traditional way of producing monoclonal antibodies is not cost-effective. The objective of this study was to find a new method for the large-scale production of monoclonal antibodies against Schistosoma japonicum (Sj).Methods A phage display antibody library for Sj was constructed. To obtain a single-chain variable fragment antibody (scFv) against Sj, the library was screened with metabolic antigens from adult Sj worms (Sj-MAg) using enzyme-linked immunosorbent assay. The soluble scFvs selected were used to detect Sj antigens in the serum of acute and chronic schistosomiasis patients.Results Six positive clones with good reactivity to Sj-MAg were obtained from the phage display antibody library of about 1.07×106 individual clones. Only two of these six clones bound specifically to Sj-MAg and were chosen for further analysis. Specific soluble anti-Sj-MAg scFvs were produced by inducing the 2 clones with isopropyl-D-thiogalactopyranoside. The characteristics of the scFvs were then determined. The results of Western blot showed that these scFvs could bind to Sj-MAg specifically and had a molecular weight of about 31 kD. When testing serum from schistosomiasis patients with one of the two specific scFvs, its sensitivity was found to be 60% and 37% in acute and chronic patients, respectively, with a specificity of 90%. When the two specific scFvs were combined, their sensitivity was found to be 75% and 57% in acute and chronic patients, respectively, with a specificity of 85%.Conclusions The results indicate that the scFvs are potentially useful for the diagnosis of schistosomiasis. The library construction also provides a useful tool for the further screening of other antibodies for both diagnostic and immunotherapeutic applications and for epitope analysis and vaccine design.

  5. Selection of a breast cancer subpopulation-specific antibody using phage display on tissue sections

    DEFF Research Database (Denmark)

    Larsen, Simon Asbjørn; Meldgaard, Theresa; Fridriksdottir, Agla J

    2015-01-01

    Breast cancer tumors are composed of heterogeneous cell populations. These populations display a high variance in morphology, growth and metastatic propensity. They respond differently to therapeutic interventions, and some may be more prone to cause recurrence. Studying individual subpopulations...... stem cells. We isolated an antibody fragment LH 7, which in immunohistochemistry experiments demonstrates specific binding to breast cancer subpopulations. The selection of antibody fragments directly on small defined areas within a larger section of malignant tissue is a novel approach by which...... has the potential to provide new insight and treatment strategies for breast cancer....

  6. The use of genomic signature distance between bacteriophages and their hosts displays evolutionary relationships and phage growth cycle determination

    Directory of Open Access Journals (Sweden)

    Regeard Christophe

    2010-07-01

    Full Text Available Abstract Background Bacteriophage classification is mainly based on morphological traits and genome characteristics combined with host information and in some cases on phage growth lifestyle. A lack of molecular tools can impede more precise studies on phylogenetic relationships or even a taxonomic classification. The use of methods to analyze genome sequences without the requirement for homology has allowed advances in classification. Results Here, we proposed to use genome sequence signature to characterize bacteriophages and to compare them to their host genome signature in order to obtain host-phage relationships and information on their lifestyle. We analyze the host-phage relationships in the four most representative groups of Caudoviridae, the dsDNA group of phages. We demonstrate that the use of phage genomic signature and its comparison with that of the host allows a grouping of phages and is also able to predict the host-phage relationships (lytic vs. temperate. Conclusions We can thus condense, in relatively simple figures, this phage information dispersed over many publications.

  7. Selection and characterization of naturally occurring single-domain (IgNAR) antibody fragments from immunized sharks by phage display.

    Science.gov (United States)

    Dooley, Helen; Flajnik, Martin F; Porter, Andrew J

    2003-09-01

    The novel immunoglobulin isotype novel antigen receptor (IgNAR) is found in cartilaginous fish and is composed of a heavy-chain homodimer that does not associate with light chains. The variable regions of IgNAR function as independent domains similar to those found in the heavy-chain immunoglobulins of Camelids. Here, we describe the successful cloning and generation of a phage-displayed, single-domain library based upon the variable domain of IgNAR. Selection of such a library generated from nurse sharks (Ginglymostoma cirratum) immunized with the model antigen hen egg-white lysozyme (HEL) enabled the successful isolation of intact antigen-specific binders matured in vivo. The selected variable domains were shown to be functionally expressed in Escherichia coli, extremely stable, and bind to antigen specifically with an affinity in the nanomolar range. This approach can therefore be considered as an alternative route for the isolation of minimal antigen-binding fragments with favorable characteristics.

  8. SINGLE CHAIN VARIABLE FRAGMENTS OF ANTIBODIES AGAINST DIPHTHERIA TOXIN B-SUBUNIT ISOLATED FROM PHAGE DISPLAY HUMAN ANTIBODY LIBRARY

    Directory of Open Access Journals (Sweden)

    Oliinyk O. S.

    2014-02-01

    Full Text Available Diphtheria toxin is an exoantigen of Corynebacterium diphtheriae that inhibits protein synthesis and kills sensitive cells. The aim of this study was to obtain human recombinant single-chain variable fragment (scFv antibodies against receptor-binding B subunit of diphtheria toxin. 12 specific clones were selected after three rounds of a phage display naїve (unimmunized human antibody library against recombinant B-subunit. scFv DNA inserts from these 12 clones were digested with MvaI, and 6 unique restriction patterns were found. Single-chain antibodies were expressed in Escherichia coli XL1-blue. The recombinant proteins were characterized by immunoblotting of bacterial extracts and detection with an anti-E-tag antibody. The toxin B-subunit-binding function of the single-chain antibody was shown by ELISA. The affinity constants for different clones were found to be from 106 to 108 М–1. Due to the fact, that these antibody fragments recognized epitopes in the receptor-binding Bsubunit of diphtheria toxin, further studies are interesting to evaluate their toxin neutralization properties and potential for therapeutic applications. Obtained scFv-antibodies can also be used for detection and investigation of biological properties of diphtheria toxin.

  9. Immune TB Antibody Phage Display Library as a Tool To Study B Cell Immunity in TB Infections.

    Science.gov (United States)

    Hamidon, Nurul Hamizah; Suraiya, Siti; Sarmiento, Maria E; Acosta, Armando; Norazmi, Mohd Nor; Lim, Theam Soon

    2017-09-07

    B cells and in particular antibodies has always played second fiddle to cellular immunity in regard to tuberculosis (TB). However, recent studies has helped position humoral immunity especially antibodies back into the foray in relation to TB immunity. Therefore, the ability to correlate the natural antibody responses of infected individuals toward TB antigens would help strengthen this concept. Phage display is an intriguing approach that can be utilized to study antibody-mediated responses against a particular infection via harvesting the B cell repertoire from infected individuals. The development of disease-specific antibody libraries or immune libraries is useful to better understand antibody-mediated immune responses against specific disease antigens. This study describes the generation of an immune single-chain variable fragment (scFv) library derived from TB-infected individuals. The immune library with an estimated diversity of 10(9) independent clones was then applied for the identification of monoclonal antibodies against Mycobacterium tuberculosis α-crystalline as a model antigen. Biopanning of the library isolated three monoclonal antibodies with unique gene usage. This strengthens the role of antibodies in TB immunity in addition to the role played by cellular immunity. The developed library can be applied against other TB antigens and aid antibody-derived TB immunity studies in the future.

  10. Development of a T7 Phage Display Library to Detect Sarcoidosis and Tuberculosis by a Panel of Novel Antigens

    Directory of Open Access Journals (Sweden)

    Harvinder Talwar

    2015-04-01

    Full Text Available Sarcoidosis is a granulomatous inflammatory disease, diagnosed through tissue biopsy of involved organs in the absence of other causes such as tuberculosis (TB. No specific serologic test is available to diagnose and differentiate sarcoidosis from TB. Using a high throughput method, we developed a T7 phage display cDNA library derived from mRNA isolated from bronchoalveolar lavage (BAL cells and leukocytes of sarcoidosis patients. This complex cDNA library was biopanned to obtain 1152 potential sarcoidosis antigens and a microarray was constructed to immunoscreen two different sets of sera from healthy controls and sarcoidosis. Meta-analysis identified 259 discriminating sarcoidosis antigens, and multivariate analysis identified 32 antigens with a sensitivity of 89% and a specificity of 83% to classify sarcoidosis from healthy controls. Additionally, interrogating the same microarray platform with sera from subjects with TB, we identified 50 clones that distinguish between TB, sarcoidosis and healthy controls. The top 10 sarcoidosis and TB specific clones were sequenced and homologies were searched in the public database revealing unique epitopes and mimotopes in each group. Here, we show for the first time that immunoscreenings of a library derived from sarcoidosis tissue differentiates between sarcoidosis and tuberculosis antigens. These novel biomarkers can improve diagnosis of sarcoidosis and TB, and may aid to develop or evaluate a TB vaccine.

  11. New Media Display Technology and Exhibition Experience

    Directory of Open Access Journals (Sweden)

    Chen-Wo Kuo

    2016-01-01

    Full Text Available As the inheritor of Chinese civilization, the National Palace Museum (hereafter referred to as the NPM, houses a world-class collection of cultural art and artifacts. Since the NPM began promoting the National Digital Archives Project in 2002, its efforts have expanded to develop a digital museum and various e-learning programs. Extending the use of digital archives to its educational and cultural industrial endeavors, the NPM has maximized the value of its exhibitions, publications, and educational programs.  In 2013, the NPM integrated creative thinking and interdisciplinary technologies, such as floating projection, augmented reality, and other sensory interactive media, to recreate the historical circumstance of 19th century East Asian maritime cultures in “Rebuilding the Tong-an Ships—New Media Art Exhibition," which opened at Huashan 1914 Creative Park and later won the Gold Award at the 2014 Digital Education Innovation Competition. Through a thorough exploration of the factors contributing to the success of "Rebuilding the Tong-an Ships," this study has isolated the two main factors of the exhibition’s popularity, namely, the compactness of the metadata and the atmosphere created by the interactive display technology.

  12. Projection display technologies for the new millennium

    Science.gov (United States)

    Kahn, Frederic J.

    2000-04-01

    Although analog CRTs continue to enable most of the world's electronic projection displays such as US consumer rear projection televisions, discrete pixel (digital) active matrix LCD and DLP reflective mirror array projectors have rapidly created large nonconsumer markets--primarily for business. Recent advances in image quality, compactness and cost effectiveness of digital projectors have the potential to revolutionize major consumer and entertainment markets as well. Digital penetration of the mainstream consumer projection TV market will begin in the hear 2000. By 2005 digital projection HDTVs could take the major share of the consumer HDTV projection market. Digital projection is expected to dominate both the consumer HDTV and the cinema market by 2010, resulting in potential shipments for all projection markets exceeding 10 M units per year. Digital projection is improving at a rate 10X faster than analog CRT projectors and 5X faster than PDP flat panels. Continued rapid improvement of digital projection is expected due to its relative immaturity and due to the wide diversity of technological improvements being pursued. Key technology enablers are the imaging panels, light sources and micro-optics. Market shares of single panel projectors, MEMs panels, LCOS panels and low T p-Si TFT LCD panel variants are expected to increase.

  13. Screening of Peptide Inhibitors of TACE from a Phage Display Random 15-Peptide Library by Recombinant TACE Ectodomain

    Institute of Scientific and Technical Information of China (English)

    Huang Wei; Yang Yuzhen; Wang Zhen; Hang Ling

    2006-01-01

    Tumor necrosis factor (TNF)-oc-converting enzyme (TACE) is the major protease responsible for processing pro-TNF-α from membrane-anchored precursors to secreted TNF-α.In the present study,a 15-peptide library was used to identify potential TACE antagonists.To obtain the recombinant TACE ectodomain and to use it as a selective molecule for the screening of peptide inhibitors of TACE,cDNA coding for the catalytic domain (T800) and full-length ectodomain (T1300) of TACE were amplified by reverse transcription-polymerase chain reaction.The expression plasmid were constructed by inserting T800/T1300 into plasmid pET-28a/c respectively and were transformed into Escherichia coli BL21 (DE3).Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) and Western blot analysis revealed that T800/T1300 were highly expressed in the form of an inclusion body induced by isopropylthiogalactoside.After Ni2+-NTA resin affinity chromatography,the purity of the recombinant T800/T1300 protein was more than 90%.T800 and T1300 proteins were used in the screening of T800/T1300-binding peptides from a phage display random 15-peptide library.Aider four rounds of biopanning,the positive phage clones were analyzed by enzyme-linked immunosorbent assay,competitive inhibition assay (ELESA),and DNA sequencing.A common amino acid sequence (TRWLVYFS RPYLVAT) was confirmed and synthesized.A synthetic peptide was shown to bind to TACE and to inhibit TNF-α release from lipopolysaccharide (LPS)-stimulated human peripheral blood mononuclear cells (PBMC) by up to 60.3%.Fluorescence-activated cell sorter (FACS) analysis revealed that the peptide mediated the accumulation of TNF-α on an LPS-stimulated PBMC surface.These results demonstrate that the TACE-binding peptide is an effective antagonist of TACE and that the deduced motif might be applied to the molecular design of anti-inflammatory drugs.

  14. Emerging Large-Screen Display Technology

    Science.gov (United States)

    1992-11-01

    1255, Santa Clara, CA. 25. Williams, R. D., and F. Garcia, 1988, "A Real Time Autostereoscopic Multiplanar 3D Display System," Society for Information...K. Miyaji, 1989, " 3D Display using Laser and Moving Screen, Japan Display 1989, Paper P3-5. 27. Sterling, R. D., R. D. TeKolste, J. M. Haggerty, T. C

  15. Advanced and tendencies in the development of display technologies

    Science.gov (United States)

    Kompanets, I. N.

    2006-06-01

    Advances and key display applications are discussed. Computer, compact mobile, TV and collective large screen displays are mentioned. Flat panel displays step on CRT devices to leave them behind in 2007. Materials, active matricies and applications of bright radiative field emission and organic LED displays are developing successively and pressing other technologies to be used in photo-cameras, cellular phones, auto-cars and avionics. Progress in flexible screens can substantially extend the display design and application soon. 3D display systems are under intensive development, and laser is an important unit in some vaiants of holographic and volumetric 3D displays. Value forecast of different display markets is presented.

  16. Phage display of the Equine arteritis virus nsp1 ZF domain and examination of its metal interactions

    DEFF Research Database (Denmark)

    Oleksiewicz, Martin B.; Snijder, E.J.; Normann, Preben

    2004-01-01

    A putative zinc finger (ZF) domain in the Equine arteritis virus (EAV) nsp 1 protein was described recently to be required for viral transcription. The nsp 1 ZF (50 aa) was expressed on the surface of M13KE gIII phage, fused to the N terminus of the phage pIII protein. To evaluate the functionality...... in the present study may have general applications in the study of other ZF domains....

  17. Designer and natural peptide toxin blockers of the KcsA potassium channel identified by phage display

    Science.gov (United States)

    Zhao, Ruiming; Dai, Hui; Mendelman, Netanel; Cuello, Luis G.; Chill, Jordan H.; Goldstein, Steve A. N.

    2015-01-01

    Peptide neurotoxins are powerful tools for research, diagnosis, and treatment of disease. Limiting broader use, most receptors lack an identified toxin that binds with high affinity and specificity. This paper describes isolation of toxins for one such orphan target, KcsA, a potassium channel that has been fundamental to delineating the structural basis for ion channel function. A phage-display strategy is presented whereby ∼1.5 million novel and natural peptides are fabricated on the scaffold present in ShK, a sea anemone type I (SAK1) toxin stabilized by three disulfide bonds. We describe two toxins selected by sorting on purified KcsA, one novel (Hui1, 34 residues) and one natural (HmK, 35 residues). Hui1 is potent, blocking single KcsA channels in planar lipid bilayers half-maximally (Ki) at 1 nM. Hui1 is also specific, inhibiting KcsA-Shaker channels in Xenopus oocytes with a Ki of 0.5 nM whereas Shaker, Kv1.2, and Kv1.3 channels are blocked over 200-fold less well. HmK is potent but promiscuous, blocking KcsA-Shaker, Shaker, Kv1.2, and Kv1.3 channels with Ki of 1–4 nM. As anticipated, one Hui1 blocks the KcsA pore and two conserved toxin residues, Lys21 and Tyr22, are essential for high-affinity binding. Unexpectedly, potassium ions traversing the channel from the inside confer voltage sensitivity to the Hui1 off-rate via Arg23, indicating that Lys21 is not in the pore. The 3D structure of Hui1 reveals a SAK1 fold, rationalizes KcsA inhibition, and validates the scaffold-based approach for isolation of high-affinity toxins for orphan receptors. PMID:26627718

  18. Identification of genes coding for B cell antigens of Mycoplasma mycoides subsp. mycoides Small Colony (MmmSC by using phage display

    Directory of Open Access Journals (Sweden)

    Vilei Edy M

    2009-10-01

    Full Text Available Abstract Background Contagious bovine pleuropneumonia (CBPP is a mycoplasmal disease caused by Mycoplasma mycoides subsp. mycoides SC (MmmSC. Since the disease is a serious problem that can affect cattle production in parts of Africa, there is a need for an effective and economical vaccine. Identifying which of the causative agent's proteins trigger potentially protective immune responses is an important step towards developing a subunit vaccine. Accordingly, the purpose of this study was to determine whether phage display combined with bioinformatics could be used to narrow the search for genes that code for potentially immunogenic proteins of MmmSC. Since the production of IgG2 and IgA are associated with a Th1 cellular immune response which is implicated in protection against CBPP, antigens which elicit these immunoglobulin subclasses may be useful in developing a subunit vaccine. Results A filamentous phage library displaying a repertoire of peptides expressed by fragments of the genome of MmmSC was constructed. It was subjected to selection using antibodies from naturally- and experimentally-infected cattle. Mycoplasmal genes were identified by matching the nucleotide sequences of DNA from immunoselected phage particles with the mycoplasmal genome. This allowed a catalogue of genes coding for the proteins that elicited an immune response to be compiled. Using this method together with computer algorithms designed to score parameters that influence surface accessibility and hence potential antigenicity, five genes (abc, gapN, glpO, lppB and ptsG were chosen to be expressed in Escherichia coli. After appropriate site-directed mutagenesis, polypeptides representing portions of each of these proteins were tested for immunoreactivity. Of these five, polypeptides representing expression products of abc and lppB were recognised on immunoblots by sera obtained from cattle during a natural outbreak of the disease. Conclusion Since phage display

  19. Application of phage display in diagnosis and therapy of cancer%噬菌体展示技术在肿瘤诊断和治疗中的应用

    Institute of Scientific and Technical Information of China (English)

    阳艳丽; 王自正

    2008-01-01

    噬菌体体内展示技术的出现,使器官靶向性多肽的筛选、器官特异性血管分子作图成为可能.更值得注意的是,通过动物体内的筛选,鉴定出了一系列肿瘤相关分子标志物的特异性多肽配体,为肿瘤的诊断、肿瘤药物的输送以及肿瘤血管性基因治疗等提供了靶向性分子.最近,人们又成功地将此技术应用于肿瘤患者,并鉴定出与肿瘤特异性结合的多肽模序.%Phage display is possible to screening targeted peptides,mapping organ-specific vascular molecule because of in vivo phage display.It is more remarkable that a variety of peptide ligands specific for tumor-associated makers is identified,which supplied targeted molecules for tumor diagnosis,delivery of tumor drugs and tumor vascular gene therapy.Recently,people succeed in applying the technology to tumor patients and identifying peptide motifs binding to tumor specifically.

  20. MimoPro: a more efficient Web-based tool for epitope prediction using phage display libraries

    Directory of Open Access Journals (Sweden)

    Guo William W

    2011-05-01

    Full Text Available Abstract Background A B-cell epitope is a group of residues on the surface of an antigen which stimulates humoral responses. Locating these epitopes on antigens is important for the purpose of effective vaccine design. In recent years, mapping affinity-selected peptides screened from a random phage display library to the native epitope has become popular in epitope prediction. These peptides, also known as mimotopes, share the similar structure and function with the corresponding native epitopes. Great effort has been made in using this similarity between such mimotopes and native epitopes in prediction, which has resulted in better outcomes than statistics-based methods can. However, it cannot maintain a high degree of satisfaction in various circumstances. Results In this study, we propose a new method that maps a group of mimotopes back to a source antigen so as to locate the interacting epitope on the antigen. The core of this method is a searching algorithm that is incorporated with both dynamic programming (DP and branch and bound (BB optimization and operated on a series of overlapping patches on the surface of a protein. These patches are then transformed to a number of graphs using an adaptable distance threshold (ADT regulated by an appropriate compactness factor (CF, a novel parameter proposed in this study. Compared with both Pep-3D-Search and PepSurf, two leading graph-based search tools, on average from the results of 18 test cases, MimoPro, the Web-based implementation of our proposed method, performed better in sensitivity, precision, and Matthews correlation coefficient (MCC than both did in epitope prediction. In addition, MimoPro is significantly faster than both Pep-3D-Search and PepSurf in processing. Conclusions Our search algorithm designed for processing well constructed graphs using an ADT regulated by CF is more sensitive and significantly faster than other graph-based approaches in epitope prediction. MimoPro is a

  1. An integrated microfluidic system for screening of phage-displayed peptides specific to colon cancer cells and colon cancer stem cells.

    Science.gov (United States)

    Che, Yu-Jui; Wu, Huei-Wen; Hung, Lien-Yu; Liu, Ching-Ann; Chang, Hwan-You; Wang, Kuan; Lee, Gwo-Bin

    2015-09-01

    Affinity reagents recognizing biomarkers specifically are essential components of clinical diagnostics and target therapeutics. However, conventional methods for screening of these reagents often have drawbacks such as large reagent consumption, the labor-intensive or time-consuming procedures, and the involvement of bulky or expensive equipment. Alternatively, microfluidic platforms could potentially automate the screening process within a shorter period of time and reduce reagent and sample consumption dramatically. It has been demonstrated recently that a subpopulation of tumor cells known as cancer stem cells possess high drug resistance and proliferation potential and are regarded as the main cause of metastasis. Therefore, a peptide that recognizes cancer stem cells and differentiates them from other cancer cells will be extremely useful in early diagnosis and target therapy. This study utilized M13 phage display technology to identify peptides that bind, respectively, to colon cancer cells and colon cancer stem cells using an integrated microfluidic system. In addition to positive selection, a negative selection process was integrated on the chip to achieve the selection of peptides of high affinity and specificity. We successfully screened three peptides specific to colon cancer cells and colon cancer stem cells, namely, HOLC-1, HOLC-2, and COLC-1, respectively, and their specificity was measured by the capture rate between target, control, and other cell lines. The capture rates are 43.40 ± 7.23%, 45.16 ± 7.12%, and 49.79 ± 5.34% for colon cancer cells and colon cancer stem cells, respectively, showing a higher specificity on target cells than on control and other cell lines. The developed technique may be promising for early diagnosis of cancer cells and target therapeutics.

  2. High-throughput sequencing enhanced phage display enables the identification of patient-specific epitope motifs in serum

    DEFF Research Database (Denmark)

    Christiansen, Anders; Kringelum, Jens Vindahl; Hansen, Christian Skjødt

    2015-01-01

    of the bioinformatic approach was demonstrated by identifying epitopes of a prominent peanut allergen, Ara h 1, in sera from patients with severe peanut allergy. The identified epitopes were confirmed by high-density peptide micro-arrays. The present study demonstrates that high-throughput sequencing can empower phage...

  3. Identification of protein kinase C inhibitory activity associated with a polypeptide isolated from a phage display system with homology to PCM-1, the pericentriolar material-1 protein.

    Science.gov (United States)

    Chakravarthy, Balu; Ménard, Michel; Brown, Leslie; Atkinson, Trevor; Whitfield, James

    2012-07-20

    We had previously identified a protein kinase C (PKC) inhibitor in murine neuroblastoma cells (Chakravarthy et al. [1]). Similar PKC inhibitory activity was also found in adult rat brain. Using polyclonal antibodies raised against the partially purified PKC inhibitor from rat brain as bait, we isolated a putative brain PKC inhibitor using a T-7 phage display system expressing human brain cDNA library. After enriching the phage population expressing the putative PKC inhibitor with four rounds of biopanning using ELISA and in vitro PKC binding assays, we identified a phage clone that expressed a product with significant PKC inhibitory activity. We have cloned and expressed this cDNA in a bacterial system and purified the recombinant protein. This polypeptide (174 amino acids) is highly homologous to a region of the 228-kDa PCM-1, the human pericentriolar material 1 protein. We have mapped this polypeptide's PKC-inhibitory domain and shown its PKC inhibitory activity in vitro. However, it will need to be determined whether the full-length PCM-1 protein possesses PKC inhibitory activity in vivo, and if so, how this might contribute to PCM-1's recently demonstrated roles in ciliogenesis and neurogenesis.

  4. Modern Display Technologies for Airborne Applications.

    Science.gov (United States)

    1983-04-01

    from the sulphonic acid group and achieved operating life of greater than 10’ cycles with over 3 years shelf-life. The switching speed was rather slow...pitch. In spite of all this ordering, however, liquid crystals are clearly liquid, flowing readily with quite low viscosity . Consequently the...solids and is the basis of most optical effects used in displays. Other physical properties, such as electrical conductivity, elasticity, viscosity

  5. Purificação e caracterização do fragmento Fab anti-digoxina obtido pela técnica de phage display.

    OpenAIRE

    2016-01-01

    A digoxina é um dos medicamentos indicados para o tratamento de falência cardíaca. Possui janela terapêutica estreita, sendo responsável por casos de intoxicação. O único antídoto disponível para a desintoxicação é o anticorpo policlonal DigiFab®, no formato Fab. O seu uso é eficaz, porém de custo elevado. Clones bacterianos produtores de fragmento Fab monoclonal anti-digoxina foram obtidos previamente pelo nosso grupo, pela técnica de phage display. Neste trabalho as variantes Fab dos 4 clo...

  6. Peptide sequences identified by phage display are immunodominant functional motifs of Pet and Pic serine proteases secreted by Escherichia coli and Shigella flexneri.

    Science.gov (United States)

    Ulises, Hernández-Chiñas; Tatiana, Gazarian; Karlen, Gazarian; Guillermo, Mendoza-Hernández; Juan, Xicohtencatl-Cortes; Carlos, Eslava

    2009-12-01

    Plasmid-encoded toxin (Pet) and protein involved in colonization (Pic), are serine protease autotransporters of Enterobacteriaceae (SPATEs) secreted by enteroaggregative Escherichia coli (EAEC), which display the GDSGSG sequence or the serine motif. Our research was directed to localize functional sites in both proteins using the phage display method. From a 12mer linear and a 7mer cysteine-constrained (C7C) libraries displayed on the M13 phage pIII protein we selected different mimotopes using IgG purified from sera of children naturally infected with EAEC producing Pet and Pic proteins, and anti-Pet and anti-Pic IgG purified from rabbits immunized with each one of these proteins. Children IgG selected a homologous group of sequences forming the consensus sequence, motif, PQPxK, and the motifs PGxI/LN and CxPDDSSxC were selected by the rabbit anti-Pet and anti-Pic IgGs, respectively. Analysis of the amino terminal region of a panel of SPATEs showed the presence in all of them of sequences matching the PGxI/LN or CxPDDSSxC motifs, and in a three-dimensional model (Modeller 9v2) designed for Pet, both these motifs were found in the globular portion of the protein, close to the protease active site GDSGSG. Antibodies induced in mice by mimotopes carrying the three aforementioned motifs were reactive with Pet, Pic, and with synthetic peptides carrying the immunogenic mimotope sequences TYPGYINHSKA and LLPQPPKLLLP, thus confirming that the peptide moiety of the selected phages induced the antibodies specific for the toxins. The antibodies induced in mice to the PGxI/LN and CxPDDSSxC mimotopes inhibited fodrin proteolysis and macrophage chemotaxis biological activities of Pet. Our results showed that we were able to generate, by a phage display procedure, mimotopes with sequence motifs PGxI/LN and CxPDDSSxC, and to identify them as functional motifs of the Pet, Pic and other SPATEs involved in their biological activities.

  7. 利用体内噬菌体展示技术筛选膀胱癌特异性结合肽%Selection of peptide specifically binding to bladder carcinoma by using phage display in vivo

    Institute of Scientific and Technical Information of China (English)

    罗俊茜; 张帆; 杨晓峰; 罗芳; 刘杰昊; 庞建智; 闫三华; 张小雷

    2015-01-01

    目的:利用体内噬菌体展示技术筛选与人膀胱移行细胞癌特异性结合的小分子多肽。方法:将人膀胱移行细胞癌细胞BIU87接种于裸鼠体内,制备膀胱癌荷瘤小鼠模型,尾静脉注射噬菌体展示环七肽库,然后筛选与膀胱移行细胞癌特异性结合的含外源多肽的噬菌体,经过3轮体内筛选后,免疫组织化学法及ELISA法鉴定单克隆噬菌体对BIU87的亲和力。提取阳性单克隆噬菌体单链DNA进行测序,并推导出外源多肽氨基酸序列,化学合成多肽、制备分子探针后采用激光扫描共聚焦显微镜术及流式细胞术鉴定多肽对膀胱癌细胞和组织的特异性。结果:3轮体内筛选后,噬菌体富集率达到4.334×102倍。免疫组化结果显示,肿瘤组织中噬菌体肽的含量随着每一轮筛选呈增长趋势,且结合逐渐增强,同时由于噬菌体经肝、肾代谢,可见肝脏结合大量非特异性的噬菌体。 ELISA结果显示,随机挑选的30个单克隆噬菌体斑中,有24个阳性噬菌体,其中10个噬菌体对BIU87有较强的亲和力,对其测序并推导出3种多肽序列,重复率最高的序列CSSPIGRHC(8/10)命名为NYZL1。化学合成FITC-C6-NYZL1,通过激光扫描共聚焦显微镜术、流式细胞术均证明多肽NYZL1可以特异性结合膀胱癌细胞。结论:利用体内噬菌体展示技术筛选出了与人膀胱移行细胞癌特异性结合的小分子多肽NYZL1,为膀胱癌早期诊断和靶向治疗提供一定的理论依据。%Objective:To screen the peptide binding to human bladder carcinoma cells specifically by using phage display technology in vivo.Methods: Nude mice were inoculated with bladder carcinoma cells BIU87 for establishing tumor-bearing mice model.The Ph.D.-C7CTM Peptide Library was injected intravenously via tail vein.Then we screened Phage containing exogenous peptides binding to bladder transitional carcinoma cells

  8. A Novel Strategy for Proteome-wide Ligand Screening Using Cross-linked Phage Matrices*

    OpenAIRE

    Qian, Chen; LIU, Jian-ning; Tang, Fengyuan; Yuan, Dawen; Guo, Zhigang; Zhang, Jing

    2010-01-01

    To find a suitable ligand from a complex antigen system is still a mission to be accomplished. Here we have explored a novel “library against proteome” panning strategy for ligand screening and antigen purification from a complex system using phage-displayed antibody technology. Human plasma proteome was targeted for phage library panning. During the process, the panning was carried out in solution, using a biotin/streptavidin beads separation system, for three rounds. Nine monoclonal phages,...

  9. Review of Display Technologies Focusing on Power Consumption

    Directory of Open Access Journals (Sweden)

    María Rodríguez Fernández

    2015-08-01

    Full Text Available This paper provides an overview of the main manufacturing technologies of displays, focusing on those with low and ultra-low levels of power consumption, which make them suitable for current societal needs. Considering the typified value obtained from the manufacturer’s specifications, four technologies—Liquid Crystal Displays, electronic paper, Organic Light-Emitting Display and Electroluminescent Displays—were selected in a first iteration. For each of them, several features, including size and brightness, were assessed in order to ascertain possible proportional relationships with the rate of consumption. To normalize the comparison between different display types, relative units such as the surface power density and the display frontal intensity efficiency were proposed. Organic light-emitting display had the best results in terms of power density for small display sizes. For larger sizes, it performs less satisfactorily than Liquid Crystal Displays in terms of energy efficiency.

  10. CERN Technology on Display in Nice

    CERN Multimedia

    2000-01-01

    Bob Dobinson (center) and Brian Martin (right) presenting technology transfer from CERN to indus exhibition in Nice. Nice in November was the scene of the EU sponsored Information Science Technologies, IST2000 exhibition, where CERN was invited to present a stand focussing on the benefits to small companies and start-ups from association with CERN and the IST program. Companies from France, Israel and the UK were featured. Network technology standards that CERN has helped to develop have been adopted by the European Space Agency for use in future space vehicles, and these too were on show. The challenges of data acquisition and data handling for the LHC are not insurmountable, but need to be approached with a combination of advanced technology and close industrial collaboration. One important aspect is that of high speed networking, an important component in both on-line and off-line computing (GRIDs). Real time data acquisition systems for the LHC require networks with high throughput, low latency, high rel...

  11. Identification of a novel aFGF-binding peptide with anti-tumor effect on breast cancer from phage display library

    Energy Technology Data Exchange (ETDEWEB)

    Dai, Xiaoyong; Cai, Cuizan [College of Pharmacy, Jinan University, Guangzhou 510632, Guangdong (China); Xiao, Fei [Department of Pharmacology, School of Medicine, Jinan University, Guangzhou 510632, Guangdong (China); Xiong, Yaoling [College of Pharmacy, Jinan University, Guangzhou 510632, Guangdong (China); Huang, Yadong; Zhang, Qihao [Department of Biopharmaceutical Research and Development Centre, Institute of Biomedicine, Jinan University, Guangzhou 510632, Guangdong (China); Xiang, Qi [College of Pharmacy, Jinan University, Guangzhou 510632, Guangdong (China); Lou, Guofeng [Department of Biopharmaceutical Research and Development Centre, Institute of Biomedicine, Jinan University, Guangzhou 510632, Guangdong (China); Lian, Mengyang [College of Pharmacy, Jinan University, Guangzhou 510632, Guangdong (China); Su, Zhijian, E-mail: tjnuszj@jnu.edu.cn [Department of Biopharmaceutical Research and Development Centre, Institute of Biomedicine, Jinan University, Guangzhou 510632, Guangdong (China); Zheng, Qing, E-mail: tzhengq@jnu.edu.cn [College of Pharmacy, Jinan University, Guangzhou 510632, Guangdong (China)

    2014-03-21

    Highlights: • A specific aFGF-binding peptide AP8 was identified from a phage display library. • AP8 could inhibit aFGF-stimulated cell proliferation in a dose-dependent manner. • AP8 arrested the cell cycle at the G0/G1 phase by suppressing Cyclin D1. • AP8 could block the activation of Erk1/2 and Akt kinase. • AP8 counteracted proliferation and cell cycle via influencing PA2G4 and PCNA. - Abstract: It has been reported that acidic fibroblast growth factor (aFGF) is expressed in breast cancer and via interactions with fibroblast growth factor receptors (FGFRs) to promote the stage and grade of the disease. Thus, aFGF/FGFRs have been considered essential targets in breast cancer therapy. We identified a specific aFGF-binding peptide (AGNWTPI, named AP8) from a phage display heptapeptide library with aFGF after four rounds of biopanning. The peptide AP8 contained two (TP) amino acids identical and showed high homology to the peptides of the 182–188 (GTPNPTL) site of high-affinity aFGF receptor FGFR1. Functional analyses indicated that AP8 specifically competed with the corresponding phage clone A8 for binding to aFGF. In addition, AP8 could inhibit aFGF-stimulated cell proliferation, arrested the cell cycle at the G0/G1 phase by increasing PA2G4 and suppressing Cyclin D1 and PCNA, and blocked the aFGF-induced activation of Erk1/2 and Akt kinase in both breast cancer cells and vascular endothelial cells. Therefore, these results indicate that peptide AP8, acting as an aFGF antagonist, is a promising therapeutic agent for the treatment of breast cancer.

  12. Antibody binding site mapping of SARS-CoV spike protein receptor-binding domain by a combination of yeast surface display and phage peptide library screening.

    Science.gov (United States)

    Zhang, Xiaoping; Wang, Jingxue; Wen, Kun; Mou, Zhirong; Zou, Liyun; Che, Xiaoyan; Ni, Bing; Wu, Yuzhang

    2009-12-01

    The receptor-binding domain (RBD) of severe acute respiratory syndrome coronavirus (SARS-CoV) spike (S) protein plays an important role in viral infection, and is a potential major neutralizing determinant. In this study, three hybridoma cell lines secreting specific monoclonal antibodies against the RBD of the S protein were generated and their exact binding sites were identified. Using yeast surface display, the binding sites of these antibodies were defined to two linear regions on the RBD: S(337-360) and S(380-399). Using these monoclonal antibodies in phage peptide library screening identified 10 distinct mimotopes 12 amino acids in length. Sequence comparison between native epitopes and these mimotopes further confirmed the binding sites, and revealed key amino acid residues involved in antibody binding. None of these antibodies could neutralize the murine leukemia virus pseudotyped expressing the SARS-CoV spike protein (MLV/SARS-CoV). However, these mAbs could be useful in the diagnosis of SARS-CoV due to their exclusive reactivity with SARS-CoV. Furthermore, this study established a feasible platform for epitope mapping. Yeast surface display combined with phage peptide library screening provides a convenient strategy for the identification of epitope peptides from certain antigenic proteins.

  13. Engineering a genetically encoded competitive inhibitor of the KEAP1-NRF2 interaction via structure-based design and phage display.

    Science.gov (United States)

    Guntas, Gurkan; Lewis, Steven M; Mulvaney, Kathleen M; Cloer, Erica W; Tripathy, Ashutosh; Lane, Thomas R; Major, Michael B; Kuhlman, Brian

    2016-01-01

    In its basal state, KEAP1 binds the transcription factor NRF2 (Kd = 5 nM) and promotes its degradation by ubiquitylation. Changes in the redox environment lead to modification of key cysteines within KEAP1, resulting in NRF2 protein accumulation and the transcription of genes important for restoring the cellular redox state. Using phage display and a computational loop grafting protocol, we engineered a monobody (R1) that is a potent competitive inhibitor of the KEAP1-NRF2 interaction. R1 bound to KEAP1 with a Kd of 300 pM and in human cells freed NRF2 from KEAP1 resulting in activation of the NRF2 promoter. Unlike cysteine-reactive small molecules that lack protein specificity, R1 is a genetically encoded, reversible inhibitor designed specifically for KEAP1. R1 should prove useful for studying the role of the KEAP1-NRF2 interaction in several disease states. The structure-based phage display strategy employed here is a general approach for engineering high-affinity binders that compete with naturally occurring interactions.

  14. Identification of antigenic epitopes of the SapA protein of Campylobacter fetus using a phage display peptide library.

    Science.gov (United States)

    Zhao, Hailing; Yu, Shenye; Liu, Huifang; Si, Wei; Wang, Chunlai; Liu, Siguo

    2012-12-01

    In this study, we immunized mice with prokaryotically expressed recombinant surface layer protein, SapA, of Campylobacter fetus, generated hybridomas secreting mouse monoclonal antibodies (mAb) targeting SapA, and purified the mAb A2D5 from mouse ascites using saturated ammonium sulfate solution. The mAb A2D5, coated onto ELISA plates, was used to screen the phage random 12-peptide library through three rounds of panning. Following panning, 15 phage clones were randomly chosen and tested for reactivity with mAb A2D5 by indirect ELISA. Single-stranded DNA from positive clones was sequenced and compared with the sequence of SapA to predict the key epitope. ELISA and/or Western blot analyses further validated that synthetic peptides and recombinant peptide mimotopes all interact with mAb A2D5. Nine of ten positive phage clones identified by screening were sequenced successfully. Seven clones shared the same sequence HYDRHNYHWWHT; one had the sequence LSKNLPLTALGN; and the final one had the sequence SGMKEPELRSYS. These three sequences shared high homology with SapA J05577 in the region GNEKDFVTKIYSIALGNTSDVDGINYW, in which the underlined amino acids may serve as key residues in the epitope. ELISA and/or Western blot analyses showed that mAb A2D5 not only interacted with the four synthetic peptide mimotopes, but also with 14 prokaryotically expressed recombinant peptide mimotopes. The mimotopes identified in this study will aid future studies into the pathological processes and immune mechanisms of the SapA protein of C. fetus. Copyright © 2012 Elsevier Ltd. All rights reserved.

  15. Biomagnetic separation of Salmonella Typhimurium with high affine and specific ligand peptides isolated by phage display technique

    Energy Technology Data Exchange (ETDEWEB)

    Steingroewer, Juliane [Institute of Food Technology and Bioprocess Engineering, Technische Universitaet Dresden, D-01062 Dresden (Germany)]. E-mail: juliane.steingroewer@tu-dresden.de; Bley, Thomas [Institute of Food Technology and Bioprocess Engineering, Technische Universitaet Dresden, D-01062 Dresden (Germany); Bergemann, Christian [Chemicell GmbH, D-10823, Berlin (Germany); Boschke, Elke [Institute of Food Technology and Bioprocess Engineering, Technische Universitaet Dresden, D-01062 Dresden (Germany)

    2007-04-15

    Analyses of food-borne pathogens are of great importance in order to minimize the health risk for customers. Thus, very sensitive and rapid detection methods are required. Current conventional culture techniques are very time consuming. Modern immunoassays and biochemical analysis also require pre-enrichment steps resulting in a turnaround time of at least 24 h. Biomagnetic separation (BMS) is a promising more rapid method. In this study we describe the isolation of high affine and specific peptides from a phage-peptide library, which combined with BMS allows the detection of Salmonella spp. with a similar sensitivity as that of immunomagnetic separation using antibodies.

  16. Technologies and solutions for data display in the operating room.

    Science.gov (United States)

    Bitterman, Noemi

    2006-06-01

    Recent advances in technology have led to the introduction of a variety of innovative devices, each with their own platform for data display, into the operating room (OR). While these innovative applications are expanding the traditional boundaries of the surgical space and enhancing treatment capabilities, the introduction of additional screens and displays is placing an ever-increasing load on the OR team. This review describes the main data display platforms currently available in ORs: computer monitors with CRT (cathode ray tube) or LCD (liquid crystal display) screens, suspended imaging displays, wearable computers (WC), auditory displays and tactile (haptic) displays. The different display platforms are evaluated according to their compatibility with the characteristics of the working environment (OR), the monitoring task, and the users (the surgical team). No single display configuration provides an ultimate solution for presenting patient data in the OR. A multi-sensory data display including visual, acoustic and haptic manipulation is suggested as a promising configuration for data display in the OR.

  17. A novel fluorescent probe: europium complex hybridized T7 phage.

    Science.gov (United States)

    Liu, Chin-Mei; Jin, Qiaoling; Sutton, April; Chen, Liaohai

    2005-01-01

    We report on the creation of a novel fluorescent probe of europium-complex hybridized T7 phage. It was made by filling a ligand-displayed T7 ghost phage with a fluorescent europium complex particle. The structure of the hybridized phage, which contains a fluorescent inorganic core surrounded by a ligand-displayed capsid shell, was confirmed by electron microscope, energy-dispersive X-ray analysis (EDX), bioassays, and fluorescence spectrometer. More importantly, as a benefit of the phage display technology, the hybridized phage has the capability to integrate an affinity reagent against virtually any target molecules. The approach provides an original method to fluorescently "tag" a bioligand and/or to "biofunctionalize" a fluorophore particle. By using other types of materials such as radioactive or magnetic particles to fill the ghost phage, we envision that the hybridized phages represent a new class of fluorescent, magnetic, or radioprobes for imaging and bioassays and could be used both in vitro and in vivo.

  18. Induction of immunity in sheep to Fasciola hepatica with mimotopes of cathepsin L selected from a phage display library.

    Science.gov (United States)

    Villa-Mancera, A; Quiroz-Romero, H; Correa, D; Ibarra, F; Reyes-Pérez, M; Reyes-Vivas, H; López-Velázquez, G; Gazarian, K; Gazarian, T; Alonso, R A

    2008-10-01

    An M13 phage random 12-mers peptide library was used to screen cathepsin L mimotopes of Fasciola hepatica and to evaluate their immunogenicity in sheep. Seven clones showed positive reactivity to a rabbit anti-cathepsin L1/L2 antiserum in ELISA, and their amino acid sequences deduced by DNA sequencing were tentatively mapped on the protein. Twenty sheep were randomly allocated into 4 groups of 5 animals each, for immunization with 1x10(14) phage particles of clones 1, 20, a mixture of 7 clones and PBS, without adjuvant at the beginning, and 4 weeks later. All groups were challenged with 300 metacercariae at week 6 and slaughtered 16 weeks later. The mean worm burdens after challenge were reduced by 47.61% and 33.91% in sheep vaccinated with clones 1 and 20, respectively; no effect was observed in animals inoculated with the clone mixture. Also, a significant reduction in worm size and burden was observed for those sheep immunized with clone 1. Animals receiving clone 20, showed a significant reduction in egg output. Immunization induced a reduction of egg viability ranging from 58.92 to 82.11%. Furthermore, vaccinated animals produced clone-specific antibodies which were boosted after challenge with metacercariae of F. hepatica.

  19. Identification of a conserved B-cell epitope on reticuloendotheliosis virus envelope protein by screening a phage-displayed random peptide library.

    Directory of Open Access Journals (Sweden)

    Mei Xue

    Full Text Available BACKGROUND: The gp90 protein of avian reticuloendotheliosis-associated virus (REV-A is an important envelope glycoprotein, which is responsible for inducing protective antibody immune responses in animals. B-cell epitopes on the gp90 protein of REV have not been well studied and reported. METHODS AND RESULTS: This study describes the identification of a linear B-cell epitope on the gp90 protein by screening a phage-displayed 12-mer random peptide library with the neutralizing monoclonal antibody (mAb A9E8 directed against the gp90. The mAb A9E8 recognized phages displaying peptides with the consensus motif SVQYHPL. Amino acid sequence of the motif exactly matched (213SVQYHPL(219 of the gp90. Further identification of the displayed B cell epitope was conducted using a set of truncated peptides expressed as GST fusion proteins and the Western blot results indicated that (213SVQYHPL(219 was the minimal determinant of the linear B cell epitope recognized by the mAb A9E8. Moreover, an eight amino acid peptide SVQYHPLA was proven to be the minimal unit of the epitope with the maximal binding activity to mAb A9E8. The REV-A-positive chicken serum reacted with the minimal linear epitopes in Western blot, revealing the importance of the eight amino acids of the epitope in antibody-epitope binding activity. Furthermore, we found that the epitope is a common motif shared among REV-A and other members of REV group. CONCLUSIONS AND SIGNIFICANCE: We identified (213SVQYHPL(219 as a gp90-specific linear B-cell epitope recognized by the neutralizing mAb A9E8. The results in this study may have potential applications in development of diagnostic techniques and epitope-based marker vaccines against REV-A and other viruses of the REV group.

  20. Large-screen display industry: market and technology trends for direct view and projection displays

    Science.gov (United States)

    Castellano, Joseph A.; Mentley, David E.

    1996-03-01

    Large screen information displays are defined as dynamic electronic displays that can be viewed by more than one person and are at least 2-feet wide. These large area displays for public viewing provide convenience, entertainment, security, and efficiency to the viewers. There are numerous uses for large screen information displays including those in advertising, transportation, traffic control, conference room presentations, computer aided design, banking, and military command/control. A noticeable characteristic of the large screen display market is the interchangeability of display types. For any given application, the user can usually choose from at least three alternative technologies, and sometimes from many more. Some display types have features that make them suitable for specific applications due to temperature, brightness, power consumption, or other such characteristic. The overall worldwide unit consumption of large screen information displays of all types and for all applications (excluding consumer TV) will increase from 401,109 units in 1995 to 655,797 units in 2002. On a unit consumption basis, applications in business and education represent the largest share of unit consumption over this time period; in 1995, this application represented 69.7% of the total. The market (value of shipments) will grow from DOL3.1 billion in 1995 to DOL3.9 billion in 2002. The market will be dominated by front LCD projectors and LCD overhead projector plates.

  1. 人源Fab抗体库的构建和抗hPRLR抗体的筛选鉴定%Screening and identification of human Fab antibody against hPRLR from large phage-display library originated from breast cancer patients

    Institute of Scientific and Technical Information of China (English)

    屈芫; 魏钦俊; 姚俊; 鲁雅洁; 王天明; 曹新; 冯振卿

    2012-01-01

    cWe aim to get specific Fab antibody against human prolactin receptor (hPRLR) from the human Fab antibody library constructed by phage display technology. Human lymphocytes were collected from peripheral blood of 24 breast cancer patients. And then the total RNA was extracted and reversely transcribed to cDNA. Genes of light and heavy chains of human antibody were amplified by RT-PCR to construct anti-hPRLR immunized human antibody library. After three rounds of panning and one round of crossed-panning with against his-hPRLR fusion protein, BSA-polypeptide (epitopes of hPRLR) and GST-hPRLR fusion protein, positive clones were chosen by Phage-ELISA and DNA sequencing. Then the positive clones were transformed into E. coli Top 10' and induced to express antibody protein. The results indicated that the human Fab phage-display library consisting of l.0×l09 clones were successfully constructed, and six clones were selected after four rounds. One of them named FabG2 expressed protein correctly. ELISA and Western blot analysis showed that FabG2 could bind hPRLR specifically. We concluded that the hPRLR specific Fab antibody selected from large phage-display library could be used as candidates for therapy agent of breast cancer which over-expresses hPRLR.%目的 构建人源Fab噬菌体抗体库,筛选抗hPRLR抗体片段并进行初步鉴定.方法 从乳腺癌患者外周血提取总RNA,通过RT-PCR扩增人抗体轻链和重链基因,构建抗hPRLR人源Fab抗体库.分别以His-hPRLR融合蛋白、BSA-hPRLR表位多肽融合蛋白和GST-hPRLR融合蛋白作为抗原包板,经过3轮循环的吸附一洗脱一扩增的筛选及1轮交叉筛选,挑单克隆用Phage-ELISA、DNA测序筛选阳性克隆,将筛选到的阳性克隆Fabc2转化至Top10’受体菌,诱导表达可溶性Fab抗体,通过Western blot和ELISA进行特异性的鉴定.结果 构建的人源Fab库容为1.0×109,4轮的筛选,获得6株能与hPRLR结合的人源抗体克隆.选取的Fabc2能够进行

  2. One-Step Recovery of scFv Clones from High-Throughput Sequencing-Based Screening of Phage Display Libraries Challenged to Cells Expressing Native Claudin-1

    Directory of Open Access Journals (Sweden)

    Emanuele Sasso

    2015-01-01

    Full Text Available Expanding the availability of monoclonal antibodies interfering with hepatitis C virus infection of hepatocytes is an active field of investigation within medical biotechnologies, to prevent graft reinfection in patients subjected to liver transplantation and to overcome resistances elicited by novel antiviral drugs. In this paper, we describe a complete pipeline for screening of phage display libraries of human scFvs against native Claudin-1, a tight-junction protein involved in hepatitis C virus infection, expressed on the cell surface of human hepatocytes. To this aim, we implemented a high-throughput sequencing approach for library screening, followed by a simple and effective strategy to recover active binder clones from enriched sublibraries. The recovered clones were successfully converted to active immunoglobulins, thus demonstrating the effectiveness of the whole procedure. This novel approach can guarantee rapid and cheap isolation of antibodies for virtually any native antigen involved in human diseases, for therapeutic and/or diagnostic applications.

  3. Development of exosome surface display technology in living human cells

    Energy Technology Data Exchange (ETDEWEB)

    Stickney, Zachary, E-mail: zstickney@scu.edu; Losacco, Joseph, E-mail: jlosacco@scu.edu; McDevitt, Sophie, E-mail: smmcdevitt@scu.edu; Zhang, Zhiwen, E-mail: zzhang@scu.edu; Lu, Biao, E-mail: blu2@scu.edu

    2016-03-25

    Surface display technology is an emerging key player in presenting functional proteins for targeted drug delivery and therapy. Although a number of technologies exist, a desirable mammalian surface display system is lacking. Exosomes are extracellular vesicles that facilitate cell–cell communication and can be engineered as nano-shuttles for cell-specific delivery. In this study, we report the development of a novel exosome surface display technology by exploiting mammalian cell secreted nano-vesicles and their trans-membrane protein tetraspanins. By constructing a set of fluorescent reporters for both the inner and outer surface display on exosomes at two selected sites of tetraspanins, we demonstrated the successful exosomal display via gene transfection and monitoring fluorescence in vivo. We subsequently validated our system by demonstrating the expected intracellular partitioning of reporter protein into sub-cellular compartments and secretion of exosomes from human HEK293 cells. Lastly, we established the stable engineered cells to harness the ability of this robust system for continuous production, secretion, and uptake of displayed exosomes with minimal impact on human cell biology. In sum, our work paved the way for potential applications of exosome, including exosome tracking and imaging, targeted drug delivery, as well as exosome-mediated vaccine and therapy.

  4. Development of exosome surface display technology in living human cells.

    Science.gov (United States)

    Stickney, Zachary; Losacco, Joseph; McDevitt, Sophie; Zhang, Zhiwen; Lu, Biao

    2016-03-25

    Surface display technology is an emerging key player in presenting functional proteins for targeted drug delivery and therapy. Although a number of technologies exist, a desirable mammalian surface display system is lacking. Exosomes are extracellular vesicles that facilitate cell-cell communication and can be engineered as nano-shuttles for cell-specific delivery. In this study, we report the development of a novel exosome surface display technology by exploiting mammalian cell secreted nano-vesicles and their trans-membrane protein tetraspanins. By constructing a set of fluorescent reporters for both the inner and outer surface display on exosomes at two selected sites of tetraspanins, we demonstrated the successful exosomal display via gene transfection and monitoring fluorescence in vivo. We subsequently validated our system by demonstrating the expected intracellular partitioning of reporter protein into sub-cellular compartments and secretion of exosomes from human HEK293 cells. Lastly, we established the stable engineered cells to harness the ability of this robust system for continuous production, secretion, and uptake of displayed exosomes with minimal impact on human cell biology. In sum, our work paved the way for potential applications of exosome, including exosome tracking and imaging, targeted drug delivery, as well as exosome-mediated vaccine and therapy.

  5. A Conserved Epitope Mapped with a Monoclonal Antibody against the VP3 Protein of Goose Parvovirus by Using Peptide Screening and Phage Display Approaches.

    Science.gov (United States)

    Li, Chenxi; Liu, Hongyu; Li, Jinzhe; Liu, Dafei; Meng, Runze; Zhang, Qingshan; Shaozhou, Wulin; Bai, Xiaofei; Zhang, Tingting; Liu, Ming; Zhang, Yun

    2016-01-01

    Waterfowl parvovirus (WPV) infection causes high mortality and morbidity in both geese (Anser anser) and Muscovy ducks (Cairina moschata), resulting in significant losses to the waterfowl industries. The VP3 protein of WPV is a major structural protein that induces neutralizing antibodies in the waterfowl. However, B-cell epitopes on the VP3 protein of WPV have not been characterized. To understand the antigenic determinants of the VP3 protein, we used the monoclonal antibody (mAb) 4A6 to screen a set of eight partially expressed overlapping peptides spanning VP3. Using western blotting and an enzyme-linked immunosorbent assay (ELISA), we localized the VP3 epitope between amino acids (aa) 57 and 112. To identify the essential epitope residues, a phage library displaying 12-mer random peptides was screened with mAb 4A6. Phage clone peptides displayed a consensus sequence of YxRFHxH that mimicked the sequence 82Y/FNRFHCH88, which corresponded to amino acid residues 82 to 88 of VP3 protein of WPVs. mAb 4A6 binding to biotinylated fragments corresponding to amino acid residues 82 to 88 of the VP3 protein verified that the 82FxRFHxH88 was the VP3 epitope and that amino acids 82F is necessary to retain maximal binding to mAb 4A6. Parvovirus-positive goose and duck sera reacted with the epitope peptide by dot blotting assay, revealing the importance of these amino acids of the epitope in antibody-epitope binding reactivity. We identified the motif FxRFHxH as a VP3-specific B-cell epitope that is recognized by the neutralizing mAb 4A6. This finding might be valuable in understanding of the antigenic topology of VP3 of WPV.

  6. Screening of scFvs against cTnI from Phage Display Antibody Library and Their Expression in E.coli Rosetta

    Institute of Scientific and Technical Information of China (English)

    WEI Jing-yan; ZHANG Han-qi; JIN Qin-han; LI Wei; LUO Gui-min; LI Shan-yu; MU Ying; ZHU Xue-jun; LIU Lei; GAO Li-zeng; SONG Da-qian; SUN Zhi-wei; YAN Gang-lin

    2005-01-01

    The single chain variable fragments of antibodies(scFvs) against eTnI were screened from the phage display antibody library by using cTnI as the target antigen. After four rounds of panning, four clones(H2, G5,A9, B9) from the phage display antibody library were verified to show higher binding affinity for cTnI by ELISA and to contain the variable region genes of the light and heavy chains of scFvs by sequencing. The variable region genes of scFvs H2 and G5 were successfully amplified by polymerase chain reactions (PCR)and cloned into expression vector pPELB and expressed as a soluble protein in E. coli Rosetta, whose expression yield was about 2% of total proteins. The expressed proteins were purified by nickel(Ni) affinity chromatography and a single band is shown in the position of 28 kDa on SDS-PAGE. The western blot analysis result verifies that the expressed scFv proteins are capable of binding with monoclonal antibodies against hexa-histidine, indicating that they are hexa-histidin-tagged aim proteins. The immunoassay demonstrates that the expressed scFv proteins are able to specifically react with cTnI molecules. The association constant (KA)values range from 1.2×104 to 1.7×105 L/mol that are correspondent to the affinities of polyclonal antibodies against cTnI from rabbits. These antibodies can be valuable reagents for the immunoassay of cTnI.

  7. Screening a phage display library for a novel FGF8b-binding peptide with anti-tumor effect on prostate cancer

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Wenhui; Chen, Xilei; Li, Tao; Li, Yanmei; Wang, Ruixue; He, Dan; Luo, Wu [Institute of Tissue Transplantation and Immunology, Jinan University, Guangzhou 510632 (China); Li, Xiaokun [Institute of Tissue Transplantation and Immunology, Jinan University, Guangzhou 510632 (China); School of Pharmaceutical Science, Wenzhou Medical College, Wenzhou 325035 (China); Wu, Xiaoping, E-mail: twxp@jnu.edu.cn [Institute of Tissue Transplantation and Immunology, Jinan University, Guangzhou 510632 (China); School of Pharmaceutical Science, Wenzhou Medical College, Wenzhou 325035 (China)

    2013-05-01

    Fibroblast growth factor 8b (FGF8b) is the major isoform of FGF8 expressed in prostate cancer and it correlates with the stage and grade of the disease. FGF8b has been considered as a potential target for prostate cancer therapy. Here we isolated 12 specific FGF8b-binding phage clones by screening a phage display heptapeptide library with FGF8b. The peptide (HSQAAVP, named as P12) corresponding to one of these clones showed high homology to the immunoglobulin-like (Ig-like) domain II(D2) of high-affinity FGF8b receptor (FGFR3c), contained 3 identical amino acids (AVP) to the authentic FGFR3 D2 sequence aa 163–169 (LLAVPAA) directly participating in ligand binding, carried the same charges as its corresponding motif (aa163–169) in FGFR3c, suggesting that P12 may have a greater potential to interrupt FGF8b binding to its receptors than other identified heptapeptides do. Functional analysis indicated that synthetic P12 peptides mediate significant inhibition of FGF8b-induced cell proliferation, arrest cell cycle at the G0/G1 phase via suppression of Cyclin D1 and PCNA, and blockade of the activations of Erk1/2 and Akt cascades in both prostate cancer cells and vascular endothelial cells. The results demonstrated that the P12 peptide acting as an FGF8b antagonist may have therapeutic potential in prostate cancer. - Highlights: ► A novel FGF8b-binding peptide P12 was isolated from a phage display library. ► The mechanisms for P12 peptide inhibiting cell proliferation were proposed. ► P12 caused cell cycle arrest at G0/G1 phase via suppression of Cyclin D1 and PCNA. ► P12 suppressed FGF8b-induced activations of Akt and MAP kinases. ► P12 acting as an FGF8b antagonist may have therapeutic potential in prostate cancer.

  8. Evolutional selection of a combinatorial phage library displaying randomly-rearranged various single domains of immunoglobulin (Ig-binding proteins (IBPs with four kinds of Ig molecules

    Directory of Open Access Journals (Sweden)

    Jia Jian-An

    2008-08-01

    Full Text Available Abstract Background Protein A, protein G and protein L are three well-defined immunoglobulin (Ig-binding proteins (IBPs, which show affinity for specific sites on Ig of mammalian hosts. Although the precise functions of these molecules are not fully understood, it is thought that they play an important role in pathogenicity of bacteria. The single domains of protein A, protein G and protein L were all demonstrated to have function to bind to Ig. Whether combinations of Ig-binding domains of various IBPs could exhibit useful novel binding is interesting. Results We used a combinatorial phage library which displayed randomly-rearranged various-peptide-linked molecules of D and A domains of protein A, designated PA(D and PA(A respectively, B2 domain of protein G (PG and B3 domain of protein L (PL for affinity selection with human IgG (hIgG, human IgM (hIgM, human IgA (hIgA and recombinant hIgG1-Fc as bait respectively. Two kinds of novel combinatorial molecules with characteristic structure of PA(A-PG and PA(A-PL were obtained in hIgG (hIgG1-Fc and hIgM (hIgA post-selection populations respectively. In addition, the linking peptides among all PA(A-PG and PA(A-PL structures was strongly selected, and showed interestingly divergent and convergent distribution. The phage binding assays and competitive inhibition experiments demonstrated that PA(A-PG and PA(A-PL combinations possess comparable binding advantages with hIgG/hIgG1-Fc and hIgM/hIgA respectively. Conclusion In this work, a combinatorial phage library displaying Ig-binding domains of protein A, protein G, or protein L joined by various random linking peptides was used to conducted evolutional selection in vitro with four kinds of Ig molecules. Two kinds of novel combinations of Ig-binding domains, PA(A-PG and PA(A-PL, were obtained, and demonstrate the novel Ig binding properties.

  9. 人源性抗乳腺癌单链抗体库的筛选与初步鉴定%Isolation and identification of single chain Fv antibodies against breast cancer from a human phage display library

    Institute of Scientific and Technical Information of China (English)

    王净; 王慧; 王超; 袁媛; 崔岩; 叶菁; 李青

    2012-01-01

    library against human breast cancer with high capacity by phage display technology. The scfv was demonstrated successfully to be expressed in E coli HB215 and have a specificity to breast cancer. Our findings may provide an alternative approach, and a basis for further studies on diagnosis and therapy of breast cancer.

  10. A phage-displayed chicken single-chain antibody fused to alkaline phosphatase detects Fusarium pathogens and their presence in cereal grains

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Zu-Quan [Molecular Biotechnology Laboratory of Triticeae Crops, Huazhong Agricultural University, Wuhan 430070 (China); Li, He-Ping [Molecular Biotechnology Laboratory of Triticeae Crops, Huazhong Agricultural University, Wuhan 430070 (China); College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Zhang, Jing-Bo [Molecular Biotechnology Laboratory of Triticeae Crops, Huazhong Agricultural University, Wuhan 430070 (China); College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Huang, Tao [Molecular Biotechnology Laboratory of Triticeae Crops, Huazhong Agricultural University, Wuhan 430070 (China); College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Liu, Jin-Long; Xue, Sheng [Molecular Biotechnology Laboratory of Triticeae Crops, Huazhong Agricultural University, Wuhan 430070 (China); Wu, Ai-Bo [Institute for Agri-food Standards and Testing Technology, Laboratory of Quality and Safety Risk Assessment for Agro-products, Ministry of Agriculture, Shanghai Academy of Agricultural Sciences, 1000 Jinqi Road, Shanghai 201403 (China); Liao, Yu-Cai, E-mail: ycliao06@yahoo.com.cn [Molecular Biotechnology Laboratory of Triticeae Crops, Huazhong Agricultural University, Wuhan 430070 (China); College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); National Center of Plant Gene Research, Wuhan 430070 (China)

    2013-02-18

    Graphical abstract: A phage-displayed chicken scFv antibody, FvSG7, binds on the surface antigen of conidiospores and the mycelia of F. verticillioides. Its fusion with alkaline phosphatase (AP) through a 218 linker displayed a 4-fold higher affinity compared with the parent scFv antibody and efficiently detected toxigenic Fusarium pathogens in cereal grains. Highlights: ► Generation of a highly reactive scFv antibody against F. verticillioides. ► Localization of the antibody binding to the surface target of F. verticillioides. ► Expression of the antibody–alkaline phosphatase (AP) fusion linked by a 218 linker. ► The antibody–AP fusion has a higher affinity than the parental antibody. ► The antibody–AP fusion detects toxigenic Fusarium pathogens in cereal grains. -- Abstract: Fusarium and its poisonous mycotoxins are distributed worldwide and are of particular interest in agriculture and food safety. A simple analytical method to detect pathogens is essential for forecasting diseases and controlling mycotoxins. This article describes a proposed method for convenient and sensitive detection of Fusarium pathogens that uses the fusion of single-chain variable fragment (scFv) and alkaline phosphatase (AP). A highly reactive scFv antibody specific to soluble cell wall-bound proteins (SCWPs) of F. verticillioides was selected from an immunized chicken phagemid library by phage display. The antibody was verified to bind on the surface of ungerminated conidiospores and mycelia of F. verticillioides. The scFv–AP fusion was constructed, and soluble expression in bacteria was confirmed. Both the antibody properties and enzymatic activity were retained, and the antigen-binding capacity of the fusion was enhanced by the addition of a linker. Surface plasmon resonance measurements confirmed that the fusion displayed 4-fold higher affinity compared with the fusion's parental scFv antibody. Immunoblot analyses showed that the fusion had good binding

  11. The technology of multiuser large display area and auto free-viewing stereoscopic display

    Science.gov (United States)

    Zhao, Tian-Qi; Zhang, He-Ling; Han, Jing

    2010-11-01

    No-glasses optical grating stereoscopic display is one of a chief development of stereoscopic display, but it is always confined by the range of stereoscopic visible and quantity of stereoscopic information and quantity of users. This research use the combination of Fresnel lens array and controllable point lights to output information of the two eyes of different users separately. Combining the technology of eyes-tracking, it can make no-glasses optical grating stereoscopic display be visible in 3D orientation range by multiuser in the condition of two-angle image sources. And it also can be visible in 360° stereoscopic overlook by one user in the condition of multi-angle image sources.

  12. 抗呼吸道合胞病毒Fab噬菌体抗体库的构建及初步筛选%Construction and preliminary panning of Fab phage display antibody library against respiratory syncytial virus

    Institute of Scientific and Technical Information of China (English)

    汪治华; 张国成; 李安茂; 周南; 陈一; 李小青; 苏字飞; 邓阳彬; 王治静

    2008-01-01

    Objective To construct a human phage display antibody library,which will help to develop new drugs and vaccines against respiratory syncytial virus (RSV) and solve many of the issues that have limited the progression and application of murine monoclonal antibodies (McAbs) in the clinic.This can provide a platform for human antibody preparation and diagnosis,prophylaxis and therapy of RSV infection in children.Methods Peripheral blood lymphocytes were isolated from 52 children with RSV infection,cDNA was synthesized from the total RNA of lymphocytes.The light and heavy chain Fd (VH-CH1)fragments of immunoglobulin gene were amplified by RT-PCR.The amplified products were cloned into phagemid vector pComb3x and the clone samples were electrotransformed into competent E.coli XL1-Blue.The transformed cells were then infected with M13K07 helper phage to yield recombinant phage antibody of Fabs.The plasmids extracted from amplified E.coil were digested with restriction endonucleases Sac 1,Xba I,Spe I and Xho I to monitor the insertion of the light or heavy chain Fd genes.RSV virions were utilized as antigens to screen Fab antibodies.Results By recombination of light and heavy chain genes,an immune Fab phage display antibody library against RSV containing 2.08×107 different clones was constructed,in which 70% clones had light chains and heavy chain Fd genes.The capacity of Fab phage antibody gene library was 1.46×107 and the titre of the original Fab antibody library was about 1.06 x 1012 pfu/mL The antibody library gained an enrichment in different degrees after the preliminary panning.Condusions Utilizing the technology of phage display,an immune Fab phage display antibody library against RSV was successfully constructed in this study,which laid a valuable experimental foundation for further study and created favorable conditions for preparing human McAbs. This may also contribute to the improvement in the diagnosis,therapy and prophylaxis of RSV infection in children

  13. Deep sequencing analysis of phage libraries using Illumina platform.

    Science.gov (United States)

    Matochko, Wadim L; Chu, Kiki; Jin, Bingjie; Lee, Sam W; Whitesides, George M; Derda, Ratmir

    2012-09-01

    This paper presents an analysis of phage-displayed libraries of peptides using Illumina. We describe steps for the preparation of short DNA fragments for deep sequencing and MatLab software for the analysis of the results. Screening of peptide libraries displayed on the surface of bacteriophage (phage display) can be used to discover peptides that bind to any target. The key step in this discovery is the analysis of peptide sequences present in the library. This analysis is usually performed by Sanger sequencing, which is labor intensive and limited to examination of a few hundred phage clones. On the other hand, Illumina deep-sequencing technology can characterize over 10(7) reads in a single run. We applied Illumina sequencing to analyze phage libraries. Using PCR, we isolated the variable regions from M13KE phage vectors from a phage display library. The PCR primers contained (i) sequences flanking the variable region, (ii) barcodes, and (iii) variable 5'-terminal region. We used this approach to examine how diversity of peptides in phage display libraries changes as a result of amplification of libraries in bacteria. Using HiSeq single-end Illumina sequencing of these fragments, we acquired over 2×10(7) reads, 57 base pairs (bp) in length. Each read contained information about the barcode (6bp), one complimentary region (12bp) and a variable region (36bp). We applied this sequencing to a model library of 10(6) unique clones and observed that amplification enriches ∼150 clones, which dominate ∼20% of the library. Deep sequencing, for the first time, characterized the collapse of diversity in phage libraries. The results suggest that screens based on repeated amplification and small-scale sequencing identify a few binding clones and miss thousands of useful clones. The deep sequencing approach described here could identify under-represented clones in phage screens. It could also be instrumental in developing new screening strategies, which can preserve

  14. Membrane insertion and assembly of epitope-tagged gp9 at the tip of the M13 phage

    Directory of Open Access Journals (Sweden)

    Kuhn Andreas

    2011-09-01

    Full Text Available Abstract Background Filamentous M13 phage extrude from infected Escherichia coli with a tip structure composed of gp7 and gp9. This tip structure is extended by the assembly of the filament composed of the major coat protein gp8. Finally, gp3 and gp6 terminate the phage structure at the proximal end. Up to now, gp3 has been the primary tool for phage display technology. However, gp7, gp8 and gp9 could also be used for phage display and these phage particles should bind to two different or more surfaces when the modified coat proteins are combined. Therefore, we tested here if the amino-terminal end of gp9 can be modified and whether the modified portion is exposed and detectable on the M13 phage particles. Results The amino-terminal region of gp9 was modified by inserting short sequences that encode antigenic epitopes. We show here that the modified gp9 proteins correctly integrate into the membrane using the membrane insertase YidC exposing the modified epitope into the periplasm. The proteins are then efficiently assembled onto the phage particles. Also extensions up to 36 amino acid residues at the amino-terminal end of gp9 did not interfere with membrane integration and phage assembly. The exposure of the antigenic tags on the phage was visualised with immunogold labelling by electron microscopy and verified by dot blotting with antibodies to the tags. Conclusions Our results suggest that gp9 at the phage tip is suitable for the phage display technology. The modified gp9 can be supplied in trans from a plasmid and fully complements M13 phage with an amber mutation in gene 9. The modified phage tip is very well accessible to antibodies.

  15. Identification of two linear B-cell epitopes from West Nile virus NS1 by screening a phage-displayed random peptide library

    Directory of Open Access Journals (Sweden)

    Qin Yong-Li

    2011-07-01

    Full Text Available Abstract Background The West Nile virus (WNV nonstructural protein 1 (NS1 is an important antigenic protein that elicits protective antibody responses in animals and can be used for the serological diagnosis of WNV infection. Although previous work has demonstrated the vital role of WNV NS1-specific antibody responses, the specific epitopes in the NS1 have not been identified. Results The present study describes the identification of two linear B-cell epitopes in WNV NS1 through screening a phage-displayed random 12-mer peptide library with two monoclonal antibodies (mAbs 3C7 and 4D1 that directed against the NS1. The mAbs 3C7 and 4D1 recognized phages displaying peptides with the consensus motifs LTATTEK and VVDGPETKEC, respectively. Exact sequences of both motifs were found in the NS1 (895LTATTEK901 and 925VVDGPETKEC934. Further identification of the displayed B cell epitopes were conducted using a set of truncated peptides expressed as MBP fusion proteins. The data indicated that 896TATTEK901 and925VVDGPETKEC934 are minimal determinants of the linear B cell epitopes recognized by the mAbs 3C7 and 4D1, respectively. Antibodies present in the serum of WNV-positive horses recognized the minimal linear epitopes in Western blot analysis, indicating that the two peptides are antigenic in horses during infection. Furthermore, we found that the epitope recognized by 3C7 is conserved only among WNV strains, whereas the epitope recognized by 4D1 is a common motif shared among WNV and other members of Japanese encephalitis virus (JEV serocomplex. Conclusions We identified TATTEK and VVDGPETKEC as NS1-specific linear B-cell epitopes recognized by the mAbs 3C7 and 4D1, respectively. The knowledge and reagents generated in this study may have potential applications in differential diagnosis and the development of epitope-based marker vaccines against WNV and other viruses of JEV serocomplex.

  16. Identification of a panel of tumor-associated antigens from breast carcinoma cell lines, solid tumors and testis cDNA libraries displayed on lambda phage

    Directory of Open Access Journals (Sweden)

    Cianfriglia Maurizio

    2004-11-01

    Full Text Available Abstract Background Tumor-associated antigens recognized by humoral effectors of the immune system are a very attractive target for human cancer diagnostics and therapy. Recent advances in molecular techniques have led to molecular definition of immunogenic tumor proteins based on their reactivity with autologous patient sera (SEREX. Methods Several high complexity phage-displayed cDNA libraries from breast carcinomas, human testis and breast carcinoma cell lines MCF-7, MDA-MB-468 were constructed. The cDNAs were expressed in the libraries as fusion to bacteriophage lambda protein D. Lambda-displayed libraries were efficiently screened with sera from patients with breast cancer. Results A panel of 21 clones representing 18 different antigens, including eight proteins of unknown function, was identified. Three of these antigens (T7-1, T11-3 and T11-9 were found to be overexpressed in tumors as compared to normal breast. A serological analysis of the 21 different antigens revealed a strong cancer-related profile for at least five clones (T6-2, T6-7, T7-1, T9-21 and T9-27. Conclusions Preliminary results indicate that patient serum reactivity against five of the antigens is associated with tumor disease. The novel T7-1 antigen, which is overexpressed in breast tumors and recognized specifically by breast cancer patient sera, is potentially useful in cancer diagnosis.

  17. 肝癌特异性噬菌体多肽的筛选和初步鉴定%Screening and preliminary identification of liver cancer specific peptide from a phage display peptide library

    Institute of Scientific and Technical Information of China (English)

    罗时敏; 臧林泉

    2009-01-01

    associated antigen ligand. Conclusion Several candidate peptide binding to liver cancer specifically have been selected by phage display technology, providing the potential uses for early diagnosis of liver cancer or targeted therapy for liver cancer.

  18. The comparison of BLyS-binding peptides from phage display library and computer-aided design on BLyS-TACI interaction.

    Science.gov (United States)

    Zhao, Yacong; Hao, Xiafei; Feng, Jiannan; Shen, Beifen; Wei, Jing; Sun, Jian

    2015-02-01

    BLyS antagonists have become the therapeutic reagents in the treatment of autoimmune disorders. BLyS binding peptides and their Fc fusion proteins may be alternative BLyS antagonists in such application. In this study, the activity of BLyS binding peptide 814 obtained from phage display library and peptide TA designed by computer-aided modeling on the interaction of BLyS-TACI was compared. In addition, to maintain the spatial conformation and stability of the peptides, human IgG1 Fc fragment was fused to peptides 814 and TA to form peptide-Fc fusion proteins, steady and innovative peptibodies. The prokaryotic expression plasmids pET30a-814-Fc and pET30a-TA-Fc for these peptibodies were acquired by genetic engineering, and confirmed by DNA sequencing. After the right plasmids were transformed into Escherichia coli BL21 (DE3), the fusion proteins were expressed and purified by protein A affinity column. As a result of competitive ELISA, peptides 814 and TA at 100μg/ml displayed 52.2% and 28.6% inhibition on the interaction of TACI-Fc with BLyS respectively. Moreover, 814-Fc and TA-Fc fusion proteins could bind to BLyS in a dosage-dependent manner as TACI-Fc did, and displayed 54.7% and 26.1% inhibition on the interaction of TACI-Fc-Myc with BLyS at 100μg/ml respectively. So 814-Fc and TA-Fc proteins had the similar bioactivity as the peptides did. Furthermore, compared with TA-Fc, 814-Fc showed two-fold inhibition effect on BLyS binding to TACI, suggesting that 814-Fc could inhibit BLyS bioactivity significantly and might serve as a potential antagonist to treat autoimmune diseases associated with BLyS overexpression.

  19. A combinatorial mutagenesis approach for functional epitope mapping on phage-displayed target antigen: application to antibodies against epidermal growth factor.

    Science.gov (United States)

    Infante, Yanelys Cabrera; Pupo, Amaury; Rojas, Gertrudis

    2014-01-01

    Although multiple different procedures to characterize the epitopes recognized by antibodies have been developed, site-directed mutagenesis remains the method of choice to define the energetic contribution of antigen residues to binding. These studies are useful to identify critical residues and to delineate functional maps of the epitopes. However, they tend to underestimate the roles of residues that are not critical for binding on their own, but contribute to the formation of the target epitope in an additive, or even cooperative, way. Mapping antigenic determinants with a diffuse energetic landscape, which establish multiple individually weak interactions with the antibody paratope, resulting in high affinity and specificity recognition of the epitope as a whole, is thus technically challenging. The current work was aimed at developing a combinatorial strategy to overcome the limitations of site-directed mutagenesis, relying on comprehensive randomization of discrete antigenic regions within phage-displayed antigen libraries. Two model antibodies recognizing epidermal growth factor were used to validate the mapping platform. Abrogation of antibody recognition due to the introduction of simultaneous replacements was able to show the involvement of particular amino acid clusters in epitope formation. The abundance of some of the original residues (or functionally equivalent amino acids sharing their physicochemical properties) among the set of mutated antigen variants selected on a given antibody highlighted their contributions and allowed delineation of a detailed functional map of the corresponding epitope. The use of the combinatorial approach could be expanded to map the interactions between other antigens/antibodies.

  20. Identification of peptides that bind hepatitis C virus envelope protein E2 and inhibit viral cellular entry from a phage-display peptide library.

    Science.gov (United States)

    Lü, Xin; Yao, Min; Zhang, Jian-Min; Yang, Jing; Lei, Ying-Feng; Huang, Xiao-Jun; Jia, Zhan-Sheng; Ma, Li; Lan, Hai-Yun; Xu, Zhi-Kai; Yin, Wen

    2014-05-01

    Hepatitis C virus (HCV) envelope protein E2 is required for the entry of HCV into cells. Viral envelope proteins interact with cell receptors in a multistep process, which may be a promising target for the development of novel antiviral agents. In this study, a heptapeptide M13 phage-display library was screened for peptides that bind specifically to prokaryotically expressed, purified truncated HCV envelope protein E2. ELISA assay was used to quantify the binding of the peptides to HCV E2 protein. Flow cytometry, quantitative reverse-transcription PCR and western blotting were used to investigate the inhibition effect of one peptide on HCV infection in hepatoma cells (Huh7.5) in vitro. Four peptides capable of binding specifically to HCV E2 protein were obtained after three rounds of biopanning. Peptide C18 (WPWHNHR), with the highest affinity for binding HCV E2 protein, was synthesized. The results showed that peptide C18 inhibited the viral infectivity of both HCV pseudotype particles (HCVpp) harboring HCV envelope glycoproteins and cell-culture produced HCV (HCVcc). Thus, this study demonstrated that peptide C18 is a potential candidate for anti-HCV therapy as a novel viral entry inhibitor.

  1. Screening of Proteins Interacting with Nonstructural 1 Protein of H5N1 Avian Influenza Virus from T7-phage Display Library

    Institute of Scientific and Technical Information of China (English)

    ZHU Chun-yu; WU Jian-guo; LIU Hong-sheng; SUN Ting-ting; ZHAO Jian; WANG Ning; ZHENG Fang-liang; AI Hai-xin; ZHU Jun-feng; WANG Xiao-ying; ZHU Ying

    2012-01-01

    Avian influenza virus(AIV) nonstructural 1(NS1) gene was amplified by real-time polymerse chain reaction(RT-PCR) and inserted into pET28a,then transformed into E.coli BL21(DE3) competent cell.With the induction of isopropyl-β-D-thiogalactoside(IPTG) and the purification of Ni-NTA column,we finally obtained purified NS1 protein.T7-phage display system was used to screen the proteins that interacted with NS1 from lung cell cDNA library.The selected positive clones were identified by DNA sequencing and analyzed by BLAST program in GeneBank.Two proteins were obtained as NS 1 binding proteins,Homo sapiens nucleolar and coiled-body phosphoprotein 1(NOLC1) and Homo sapiens similar to colon cancer-associated antigen.By co-immunoprecipitation and other methods,Homo sapiens NOLC1 was found to interact with the NS1 protein,the results would provide the basis for further studying biological function of NS1 protein.

  2. Comprehensive mapping of functional epitopes on dengue virus glycoprotein E DIII for binding to broadly neutralizing antibodies 4E11 and 4E5A by phage display.

    Science.gov (United States)

    Frei, Julia C; Kielian, Margaret; Lai, Jonathan R

    2015-11-01

    Here we investigated the binding of Dengue virus envelope glycoprotein domain III (DIII) by two broadly neutralizing antibodies (bNAbs), 4E11 and 4E5A. There are four serotypes of Dengue virus (DENV-1 to -4), whose DIII sequences vary by up to 49%. We used combinatorial alanine scanning mutagenesis, a phage display approach, to map functional epitopes (those residues that contribute most significantly to the energetics of antibody-antigen interaction) on these four serotypes. Our results showed that 4E11, which binds strongly to DENV-1, -2, and -3, and moderately to DENV-4, recognized a common conserved core functional epitope involving DIII residues K310, L/I387, L389, and W391. There were also unique recognition features for each serotype, suggesting that 4E11 has flexible recognition requirements. Similar scanning studies for the related bNAb 4E5A, which binds more tightly to DENV-4, identified broader functional epitopes on DENV-1. These results provide useful information for immunogen and therapeutic antibody design.

  3. Homology modelling and bivalent single-chain Fv construction of anti-HepG2 single-chain immunoglobulin Fv fragments from a phage display library

    Indian Academy of Sciences (India)

    Ming Ni; Bing Yu; Y U Huang; Zhenjie Tang; Ping Lei; Xin Shen; Wei Xin; Huifen Zhu; Guanxin Shen

    2008-12-01

    We prepared single-chain immunoglobulin Fv fragments (scFv) SLH10 specific for the HepG2 cell line after biopanning from a large human-naïve phage display library (Griffin. 1 Library). The three-dimensional (3D) structure of SLH10 was modelled by the Insight II molecule simulation software. The structure was refined using the molecular dynamics method. The structures with the least steric clashes and lowest energy were determined finally. The optimized structures of heavy (VH) and light (VL) variable chains of SLH10 scFv were obtained. Then SLH10 bivalent single-chain Fv (BsFv) was constructed that would be suitable for high-affinity targeting. SLH10 BsFv was generated by linking scFvs together and identified by sequencing. Its expression products were confirmed by western blot analysis. The relative molecular masses of scFv and BsFv were approximately 30 kDa and 60 kDa, respectively. Flow cytometry revealed that SLH10 BsFv bound the selected cell lines with greater signal intensity than the parental scFv. The improved antigen binding of SLH10 BsFv may be useful for immunodiagnostics or targeted gene therapy for liver cancer.

  4. A modified protocol for RNA isolation from high polysaccharide containing Cupressus arizonica pollen. Applications for RT-PCR and phage display library construction.

    Science.gov (United States)

    Pico de Coaña, Yago; Parody, Nuria; Fernández-Caldas, Enrique; Alonso, Carlos

    2010-02-01

    RNA isolation is the first step in the study of gene expression and recombinant protein production. However, the isolation of high quantity and high-quality RNA from tissues containing large amounts of polysaccharides has proven to be a difficult process. Cupressus arizonica pollen, in addition to containing high polysaccharide levels, is a challenging starting material for RNA isolation due to the roughness of the pollen grain's walls. Here, we describe an improved technique for RNA isolation from C. arizonica pollen grains. The protocol includes a special disruption and homogenization process as well as a two-step modified RNA isolation technique which consists of an acid phenol extraction followed by a final cleanup using a commercial kit. Resulting RNA proved to be free of contaminants as determined by UV spectrophotometry. The quality of the RNA was analyzed on a bioanalyzer and showed visible 25S and 18S bands. This RNA was successfully used in downstream applications such as RT-PCR and phage display library construction.

  5. A phage display selected 7-mer peptide inhibitor of the Tannerella forsythia metalloprotease-like enzyme Karilysin can be truncated to Ser-Trp-Phe-Pro.

    Directory of Open Access Journals (Sweden)

    Peter Durand Skottrup

    Full Text Available Tannerella forsythia is a gram-negative bacteria, which is strongly associated with the development of periodontal disease. Karilysin is a newly identified metalloprotease-like enzyme, that is secreted from T. forsythia. Karilysin modulates the host immune response and is therefore considered a likely drug target. In this study peptides were selected towards the catalytic domain from Karilysin (Kly18 by phage display. The peptides were linear with low micromolar binding affinities. The two best binders (peptide14 and peptide15, shared the consensus sequence XWFPXXXGGG. A peptide15 fusion with Maltose Binding protein (MBP was produced with peptide15 fused to the N-terminus of MBP. The peptide15-MBP was expressed in E. coli and the purified fusion-protein was used to verify Kly18 specific binding. Chemically synthesised peptide15 (SWFPLRSGGG could inhibit the enzymatic activity of both Kly18 and intact Karilysin (Kly48. Furthermore, peptide15 could slow down the autoprocessing of intact Kly48 to Kly18. The WFP motif was important for inhibition and a truncation study further demonstrated that the N-terminal serine was also essential for Kly18 inhibition. The SWFP peptide had a Ki value in the low micromolar range, which was similar to the intact peptide15. In conclusion SWFP is the first reported inhibitor of Karilysin and can be used as a valuable tool in structure-function studies of Karilysin.

  6. A phage display selected 7-mer peptide inhibitor of the Tannerella forsythia metalloprotease-like enzyme Karilysin can be truncated to Ser-Trp-Phe-Pro.

    Science.gov (United States)

    Skottrup, Peter Durand; Sørensen, Grete; Ksiazek, Miroslaw; Potempa, Jan; Riise, Erik

    2012-01-01

    Tannerella forsythia is a gram-negative bacteria, which is strongly associated with the development of periodontal disease. Karilysin is a newly identified metalloprotease-like enzyme, that is secreted from T. forsythia. Karilysin modulates the host immune response and is therefore considered a likely drug target. In this study peptides were selected towards the catalytic domain from Karilysin (Kly18) by phage display. The peptides were linear with low micromolar binding affinities. The two best binders (peptide14 and peptide15), shared the consensus sequence XWFPXXXGGG. A peptide15 fusion with Maltose Binding protein (MBP) was produced with peptide15 fused to the N-terminus of MBP. The peptide15-MBP was expressed in E. coli and the purified fusion-protein was used to verify Kly18 specific binding. Chemically synthesised peptide15 (SWFPLRSGGG) could inhibit the enzymatic activity of both Kly18 and intact Karilysin (Kly48). Furthermore, peptide15 could slow down the autoprocessing of intact Kly48 to Kly18. The WFP motif was important for inhibition and a truncation study further demonstrated that the N-terminal serine was also essential for Kly18 inhibition. The SWFP peptide had a Ki value in the low micromolar range, which was similar to the intact peptide15. In conclusion SWFP is the first reported inhibitor of Karilysin and can be used as a valuable tool in structure-function studies of Karilysin.

  7. Toward a Code for the Interactions of Zinc Fingers with DNA: Selection of Randomized Fingers Displayed on Phage

    Science.gov (United States)

    Choo, Yen; Klug, Aaron

    1994-11-01

    We have used two selection techniques to study sequence-specific DNA recognition by the zinc finger, a small, modular DNA-binding minidomain. We have chosen zinc fingers because they bind as independent modules and so can be linked together in a peptide designed to bind a predetermined DNA site. In this paper, we describe how a library of zinc fingers displayed on the surface of bacteriophage enables selection of fingers capable of binding to given DNA triplets. The amino acid sequences of selected fingers which bind the same triplet are compared to examine how sequence-specific DNA recognition occurs. Our results can be rationalized in terms of coded interactions between zinc fingers and DNA, involving base contacts from a few α-helical positions. In the paper following this one, we describe a complementary technique which confirms the identity of amino acids capable of DNA sequence discrimination from these positions.

  8. Phage display reveals multiple contact sites between FhuA, an outer membrane receptor of Escherichia coli, and TonB.

    Science.gov (United States)

    Carter, David M; Gagnon, Jean-Nicolas; Damlaj, Moussab; Mandava, Suneeta; Makowski, Lee; Rodi, Diane J; Pawelek, Peter D; Coulton, James W

    2006-03-17

    The ferric hydroxamate uptake receptor FhuA from Escherichia coli transports siderophores across the outer membrane (OM). TonB-ExbB-ExbD transduces energy from the cytoplasmic membrane to the OM by contacts between TonB and OM receptors that contain the Ton box, a consensus sequence near the N terminus. Although the Ton box is a region of known contact between OM receptors and TonB, our biophysical studies established that TonB binds to FhuA through multiple regions of interaction. Panning of phage-displayed random peptide libraries (Ph.D.-12, Ph.D.-C7C) against TonB identified peptide sequences that specifically interact with TonB. Analyses of these sequences using the Receptor Ligand Contacts (RELIC) suite of programs revealed clusters of multiply aligned peptides that mapped to FhuA. These clusters localized to a continuous periplasm-accessible surface: Ton box/switch helix; cork domain/beta1 strand; and periplasmic turn 8. Guided by such matches, synthetic oligonucleotides corresponding to DNA sequences identical to fhuA were fused to malE; peptides corresponding to the above regions were displayed at the N terminus of E.coli maltose-binding protein (MBP). Purified FhuA peptides fused to MBP bound specifically to TonB by ELISA. Furthermore, they competed with ligand-loaded FhuA for binding to TonB. RELIC also identified clusters of multiply aligned peptides corresponding to the Ton box regions in BtuB, FepA, and FecA; to periplasmic turn 8 in BtuB and FecA; and to periplasmic turns 1 and 2 in FepA. These experimental outcomes identify specific molecular contacts made between TonB and OM receptors that extend beyond the well-characterized Ton box.

  9. Biopanning and characterization of peptides with Fe3O4 nanoparticles-binding capability via phage display random peptide library technique.

    Science.gov (United States)

    You, Fei; Yin, Guangfu; Pu, Ximing; Li, Yucan; Hu, Yang; Huang, Zhongbin; Liao, Xiaoming; Yao, Yadong; Chen, Xianchun

    2016-05-01

    Functionalization of inorganic nanoparticles (NPs) play an important role in biomedical applications. A proper functionalization of NPs can improve biocompatibility, avoid a loss of bioactivity, and further endow NPs with unique performances. Modification with vairous specific binding biomolecules from random biological libraries has been explored. In this work, two 7-mer peptides with sequences of HYIDFRW and TVNFKLY were selected from a phage display random peptide library by using ferromagnetic NPs as targets, and were verified to display strong binding affinity to Fe3O4 NPs. Fourier transform infrared spectrometry, fluorescence microscopy, thermal analysis and X-ray photoelectron spectroscopy confirmed the presence of peptides on the surface of Fe3O4 NPs. Sequence analyses revealed that the probable binding mechanism between the peptide and Fe3O4 NPs might be driven by Pearson hard acid-hard base specific interaction and hydrogen bonds, accompanied with hydrophilic interactions and non-specific electrostatic attractions. The cell viability assay indicated a good cytocompatibility of peptide-bound Fe3O4 NPs. Furthermore, TVNFKLY peptide and an ovarian tumor cell A2780 specific binding peptide (QQTNWSL) were conjugated to afford a liner 14-mer peptide (QQTNWSLTVNFKLY). The binding and targeting studies showed that 14-mer peptide was able to retain both the strong binding ability to Fe3O4 NPs and the specific binding ability to A2780 cells. The results suggested that the Fe3O4-binding peptides would be of great potential in the functionalization of Fe3O4 NPs for the tumor-targeted drug delivery and magnetic hyperthermia.

  10. MicroDisplay technology: from eyeglass-mounted displays to portable projectors to the Dick Tracy video phone

    Science.gov (United States)

    Alvelda, Phillip

    1997-06-01

    MicroDisplay devices are based on a combination of technologies ranging from the extreme integration capability of conventionally fabricated CMOS active-matrix liquid crystal display substrates to proprietary tunable color- filter technology, to optical distortion correction technology for lens-system compensation. All of these technologies were devised to create a line of application- specific integrated circuit single-chip display devices with integrated computing, memory, and communication circuitry. Next-generation portable communication, computer, and consumer electronic devices such as truly portable monitor and TV projectors, eyeglass-clip-on virtual displays, pagers and personal communication services hand-sets, and wristwatch-mounted video phones are all target markets for MicroDisplay technology.

  11. 肺癌特异性结合多肽的体外筛选和鉴定%Screening and identifying the peptide specifically binding to lung cancer by using phage display in vitro

    Institute of Scientific and Technical Information of China (English)

    潘雪刁; 何冰; 王桂香; 刘金泳; 曹成明; 臧林泉

    2013-01-01

    目的 应用噬菌体随机肽库技术筛选出与肺癌细胞特异性结合的多肽.方法 以人肺癌细胞NCI-H1299为靶细胞,人胚肺细胞MRC-5为吸附细胞,对噬菌体随机12肽库进行3轮全细胞减性筛选后,随机挑取噬菌体克隆进行ELISA鉴定;对亲和力较高的阳性克隆进行DNA测序并翻译为氨基酸序列;化学合成异硫氰酸荧光素标记的多肽(FITC-ZS-5),采用细胞和组织免疫荧光法鉴定FITC-ZS-5与肺癌细胞的亲和力及特异性.结果 通过3轮减性筛选后,与NCI-H1299细胞结合的噬菌体克隆得到有效的富集:ELISA结果显示5号克隆对CI-H1299细胞亲和力最高,将其命名为Phage ZS-5:测序结果显示Phage ZS-5所表达的多肽序列在国内外均未见报道,细胞及组织免疫荧光实验结果显示FITC-ZS-5 对肺癌细胞及组织具有较高的亲和力和特异性.结论 应用噬菌体随机肽库技术筛选到肺癌靶向性多肽ZS-5,为肺癌的靶向治疗和诊断奠定基础.%Aim In vitro phage display was used to screen and identify a polypeptide specifically targeting lung cancer cells. Methods The lung cancer NCI-HI 299 cell line and the normal lung MRC-5 cell line were used for the subtractive screening in vitro with a phage display-12 peptide library at 37℃. After three rounds of panning, a group of phage clones specifically binding to the NCI-H1299 cells were obtained, then the affinity of these clones binding to the targeted cells was studied by cell-based ELISA. The phage clones with high affinity was sequenced, thus the amino acid sequence was deduced and the peptide was synthesized and identified by immunofluorescence. Results After three rounds of panning, there was an obvious enrichment for the phage clones specifically binding to the NCI-H1299 cells. A phage clone named phage ZS-5 with high specificity and affinity to NCI-H1299 cells was identified by a cell-based ELISA. More importantly, the synthetic FITC-labeled peptide ZS-5, which

  12. NOVEL AMYLOID-BETA SPECIFIC scFv and VH ANTIBODY FRAGMENTS FROM HUMAN AND MOUSE PHAGE DISPLAY ANTIBODY LIBRARIES

    Science.gov (United States)

    Medecigo, M.; Manoutcharian, K.; Vasilevko, V.; Govezensky, T.; Munguia, M. E.; Becerril, B.; Luz-Madrigal, A.; Vaca, L.; Cribbs, D. H.; Gevorkian, G.

    2010-01-01

    Anti-amyloid immunotherapy has been proposed as an appropriate therapeutic approach for Alzheimer’s disease (AD). Significant efforts have been made towards the generation and assessment of antibody-based reagents capable of preventing and clearing amyloid aggregates as well as preventing their synaptotoxic effects. In this study, we selected a novel set of human anti-amyloid-beta peptide 1-42 (Aβ1-42) recombinant monoclonal antibodies in a single chain fragment variable (scFv) and a single domain (VH) formats. We demonstrated that these antibody fragments recognize in a specific manner amyloid beta deposits in APP/Tg mouse brains, inhibit toxicity of oligomeric Aβ1-42 in neuroblastoma cell cultures in a concentration-dependently manner and reduced amyloid deposits in APP/Tg2576 mice after intracranial administration. These antibody fragments recognize epitopes in the middle/C-terminus region of Aβ, which makes them strong therapeutic candidates due to the fact that most of the Aβ species found in the brains of AD patients display extensive N-terminus truncations/modifications. PMID:20451261

  13. Phage therapy pharmacology phage cocktails.

    Science.gov (United States)

    Chan, Benjamin K; Abedon, Stephen T

    2012-01-01

    Phage therapy is the clinical or veterinary application of bacterial viruses (bacteriophages) as antibacterial "drugs." More generally, phages can be used as biocontrol agents against plant as well as foodborne pathogens. In this chapter, we consider the therapeutic use of phage cocktails, which is the combining of two or more phage types to produce more pharmacologically diverse formulations. The primary motivation for the use of cocktails is their broader spectra of activity in comparison to individual phage isolates: they can impact either more bacterial types or achieve effectiveness under a greater diversity of conditions. The combining of phages can also facilitate better targeting of multiple strains making up individual bacterial species or covering multiple species that might be responsible for similar disease states, in general providing, relative to individual phage isolates, a greater potential for presumptive or empirical treatment. Contrasting the use of phage banks, or even phage isolation against specific etiologies that have been obtained directly from patients under treatment, here we consider the utility as well as potential shortcomings associated with the use of phage cocktails as therapeutic antibacterial agents.

  14. Application of BRED technology to construct recombinant D29 reporter phage expressing EGFP.

    Science.gov (United States)

    da Silva, Joas L; Piuri, Mariana; Broussard, Gregory; Marinelli, Laura J; Bastos, Gisele M; Hirata, Rosario D C; Hatfull, Graham F; Hirata, Mario H

    2013-07-01

    Bacteriophage Recombineering of Electroporated DNA (BRED) has been described for construction of gene deletion and point mutations in mycobacteriophages. Using BRED, we inserted a Phsp60-egfp cassette (1143 bp) into the mycobacteriophage D29 genome to construct a new reporter phage, which was used for detection of mycobacterial cells. The cassette was successfully inserted and recombinant mycobacteriophage purified. DNA sequencing of the cassette did not show any mutations even after several phage generations. Mycobacterium smegmatis mc(2) 155 cells were infected with D29::Phsp60-egfp (MOI of 10) and evaluated for EGFP expression by microscopy. Fluorescence was observed at around 2 h after infection, but dissipated in later times because of cell lysis. We attempted to construct a lysis-defective mutant by deleting the lysA gene, although we were unable to purify the mutant to homogeneity even with complementation. These observations demonstrate the ability of BRED to insert c. 1 kbp-sized DNA segments into mycobacteriophage genomes as a strategy for constructing new diagnostic reporter phages.

  15. The isolation of novel phage display-derived human recombinant antibodies against CCR5, the major co-receptor of HIV.

    Science.gov (United States)

    Shimoni, Moria; Herschhorn, Alon; Britan-Rosich, Yelena; Kotler, Moshe; Benhar, Itai; Hizi, Amnon

    2013-08-01

    Selecting for antibodies against specific cell-surface proteins is a difficult task due to many unrelated proteins that are expressed on the cell surface. Here, we describe a method to screen antibody-presenting phage libraries against native cell-surface proteins. We applied this method to isolate antibodies that selectively recognize CCR5, which is the major co-receptor for HIV entry (consequently, playing a pivotal role in HIV transmission and pathogenesis). We employed a phage screening strategy by using cells that co-express GFP and CCR5, along with an excess of control cells that do not express these proteins (and are otherwise identical to the CCR5-expressing cells). These control cells are intended to remove most of the phages that bind the cells nonspecifically; thus leading to an enrichment of the phages presenting anti-CCR5-specific antibodies. Subsequently, the CCR5-presenting cells were quantitatively sorted by flow cytometry, and the bound phages were eluted, amplified, and used for further successive selection rounds. Several different clones of human single-chain Fv antibodies that interact with CCR5-expressing cells were identified. The most specific monoclonal antibody was converted to a full-length IgG and bound the second extracellular loop of CCR5. The experimental approach presented herein for screening for CCR5-specific antibodies can be applicable to screen antibody-presenting phage libraries against any cell-surface expressed protein of interest.

  16. Production and radioimmunoimaging of novel fully human phage display recombinant antibodies and growth inhibition of lung adenocarcinoma cell line overexpressing Prx I.

    Science.gov (United States)

    Luo, Yi; Pang, Hua; Li, Shujie; Cao, Hui; Peng, Zhiping; Fan, Chunbo; Li, Shaolin

    2009-07-01

    The Peroxiredoxin I (Prx I) is a member of the Peroxiredoxin family, which is overexpressed in many diverse tumor types and is an anti-apoptosis protein for tumor cell proliferation and survival. Therapeutic strategies targeting the Prx I may therefore be effective broad-spectrum anticancer agents. We constructed a phage display single-chain variable fragment (scFv) antibody library and sieve out the fully human, lung adenocarcinoma-sepcific monoclonal antibodies. The selection on Prx I was performed using above-mentioned lung adenocarcinoma-sepcific monoclonal antibodies with high affinity to Prx I overexpressing lung adenocarcinoma cells. The candidate scFv sequences, based on enzyme-linked immunosorbent assay (ELISA) screening data, were chosen for soluble expression, and a 30 kDa band was observed on polyacrylamide gel electrophoresis as predicted. The purified antibodies were characterized by immunoblotting and showed high specificity to Prx I-overexpressing lung adenocarcinoma cells A549. Radioimmunoimaging was taken to evaluate specificity and distribution of antibodies in vivo. The radiolocalization index (RI) of tumor/serum and tumor/muscle gradually increased, reaching its peak (4.06 +/- 0.13 and 5.17 +/- 0.97, respectively) at 48 h postadministration. Single photon emission computed tomography (SPECT) imaging showed the radioactivity was aggregated in tumor locations and tumor imaging was clearly observed. The internalized scFv resulted in antibody-mediated cell apoptosis and downregulation of Prx I expression. These results demonstrate that the scFv possesses strong antitumor activity on lung adenocarcinoma and may therefore be an effective therapeutic candidate for the treatment of cancers that are dependent on Prx I for growth and survival.

  17. Cyclic RGD peptide incorporation on phage major coat proteins for improved internalization by HeLa cells.

    Science.gov (United States)

    Choi, Dong Shin; Jin, Hyo-Eon; Yoo, So Young; Lee, Seung-Wuk

    2014-02-19

    Delivering therapeutic materials or imaging reagents into specific tumor tissues is critically important for development of novel cancer therapeutics and diagnostics. Genetically engineered phages possess promising structural features to develop cancer therapeutic materials. For cancer targeting purposes, we developed a novel engineered phage that expressed cyclic RGD (cRGD) peptides on the pVIII major coat protein using recombinant DNA technology. Using a type 88 phage engineering approach, which inserts a new gene to express additional major coat protein in the noncoding region of the phage genome, we incorporated an additional pVIII major coat protein with relatively bulky cRGD and assembled heterogeneous major coat proteins on the F88.4 phage surfaces. With IPTG control, we could tune different numbers of cRGD peptide displayed on the phage particles up to 140 copies. The resulting phage with cRGD on the recombinant pVIII protein exhibited enhanced internalization efficiency into HeLa cells in a ligand density and conformational structure dependent manner when comparing with the M13 phages modified with either linear RGD on pVIII or cRGD on pIII. Our cRGD peptide engineered phage could be useful for cancer therapy or diagnostic purposes after further modifying the phage with drug molecules or contrast reagents in the future.

  18. Amplification and polyclonal antibody preparation of M13 phage display library%M13噬菌体肽库扩增及兔抗血清制备

    Institute of Scientific and Technical Information of China (English)

    任晓峰; 马晓微

    2013-01-01

    为制备M13噬菌体多克隆抗体,将噬菌体十二肽原始文库进行大量扩增,作为免疫原制备兔抗M13的多克隆抗体血清并进行间接ELISA鉴定.结果表明,该多抗效价达1∶1 048 576,说明此多抗可与扩增噬菌体文库发生很好的抗原抗体反应.将制备的噬菌体M13多克隆抗体与商业化M13抗体水平比较,制备多抗与商业化M13抗体效果相当.本研究成功制备兔抗M13噬菌体多克隆抗体,为深入研究噬菌体展示技术提供了材料.%In order to generate polyclonal antibodies of the M13 Phage,one M13 of Ph..D.-12TM Phage Display Peptide Library was amplified and used to immunize a rabbit to generate polyclonal antibody.Indirect ELISA analysis showed that the titer of the polyclonal antibody was approximately 1∶1 048 576,showing that the anti-M13 antibody had a high titer.Compared this polyclonal antibody with a Rabbit polyclonal antibody (Rb pAb) to M13 Bacteriophage Coat Proteins (commercialization),the binding activity of the produced polyclonal antibody to the identical target was as good as that of the Rb pAb to M13 Bacteriophage Coat Proteins.In the study,polyclonal antibody to this M13 of phage library was successfully generated and such phage polyclonal antibody is important material for functional analysis with phage.

  19. Multipurpose Panel Display Device Investigation. [technology assessment and product development

    Science.gov (United States)

    Sliwa, R.

    1977-01-01

    A multipurpose panel was developed to provide a flexible control and a LED display panel with easily changeable nomenclature for use in applications where panel space is limited, but where a number of similar subsystems must be controlled, or where basic panel nomenclature and functions must be changed rapidly, as in the case of between mission changes of space shuttle payloads. In the first application, panel area limitations are overcome by time sharing a central control panel among several subsystems. In the latter case, entire control panel changes are effected by simply replacing a memory module, thereby reducing the extent of installation and checkout procedures between missions. Several types of control technologies (other than LED's) which show potential in meeting criteria for overcoming limitations of the panel are assessed.

  20. The phage lytic proteins from the Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88 display multiple active catalytic domains and do not trigger staphylococcal resistance.

    Directory of Open Access Journals (Sweden)

    Lorena Rodríguez-Rubio

    Full Text Available The increase in antibiotic resistance world-wide revitalized the interest in the use of phage lysins to combat pathogenic bacteria. In this work, we analyzed the specific cleavage sites on the staphylococcal peptidoglycan produced by three phage lytic proteins. The investigated cell wall lytic enzymes were the endolysin LysH5 derived from the S. aureus bacteriophage vB_SauS-phi-IPLA88 (phi-IPLA88 and two fusion proteins between lysostaphin and the virion-associated peptidoglycan hydrolase HydH5 (HydH5SH3b and HydH5Lyso. We determined that all catalytic domains present in these proteins were active. Additionally, we tested for the emergence of resistant Staphylococcus aureus to any of the three phage lytic proteins constructs. Resistant S. aureus could not be identified after 10 cycles of bacterial exposure to phage lytic proteins either in liquid or plate cultures. However, a quick increase in lysostaphin resistance (up to 1000-fold in liquid culture was observed. The lack of resistant development supports the use of phage lytic proteins as future therapeutics to treat staphylococcal infections.

  1. DNA Libraries for the Construction of Phage Libraries: Statistical and Structural Requirements and Synthetic Methods

    Directory of Open Access Journals (Sweden)

    Thomas Lindner

    2011-02-01

    Full Text Available Peptide-based molecular probes identified by bacteriophage (phage display technology expand the peptide repertoire for in vivo diagnosis and therapy of cancer. Numerous peptides that bind cancer-associated antigens have been discovered by panning phage libraries. However, until now only few of the peptides selected by phage display have entered clinical applications. The success of phage derived peptides essentially depends on the quality of the library screened. This review summarizes the methods to achieve highly homogenous libraries that cover a maximal sequence space. Biochemical and chemical strategies for the synthesis of DNA libraries and the techniques for their integration into the viral genome are discussed in detail. A focus is set on the methods that enable the exclusion of disturbing sequences. In addition, the parameters that define the variability, the minimal numbers of copies per library and the use of alternating panning cycles to avoid the loss of selected hits are evaluated.

  2. DNA libraries for the construction of phage libraries: statistical and structural requirements and synthetic methods.

    Science.gov (United States)

    Lindner, Thomas; Kolmar, Harald; Haberkorn, Uwe; Mier, Walter

    2011-02-15

    Peptide-based molecular probes identified by bacteriophage (phage) display technology expand the peptide repertoire for in vivo diagnosis and therapy of cancer. Numerous peptides that bind cancer-associated antigens have been discovered by panning phage libraries. However, until now only few of the peptides selected by phage display have entered clinical applications. The success of phage derived peptides essentially depends on the quality of the library screened. This review summarizes the methods to achieve highly homogenous libraries that cover a maximal sequence space. Biochemical and chemical strategies for the synthesis of DNA libraries and the techniques for their integration into the viral genome are discussed in detail. A focus is set on the methods that enable the exclusion of disturbing sequences. In addition, the parameters that define the variability, the minimal numbers of copies per library and the use of alternating panning cycles to avoid the loss of selected hits are evaluated.

  3. Photoaligning and photopatterning technology: applications in displays and photonics

    Science.gov (United States)

    Chigrinov, Vladimir

    2016-03-01

    The advantages of LC photoalignment technology in comparison with common "rubbing" alignment methods tend to the continuation of the research in this field. Almost all the criteria of perfect LC alignment are met in case of azo-dye layers. Nowadays azo-dye alignment materials can be already used in LCD manufacturing, e.g. for the alignment of monomers in LCP films for new generations of photonics and optics devices. Recently the new application of photoaligned technology for the tunable LC lenses with a variable focal distance was proposed. New optically rewritable (ORW) liquid crystal display and photonics devices with a light controllable structure may include LC E-paper screens, LC lenses with a variable focal distance etc. Fast ferroelectric liquid crystal devices (FLCD) are achieved through the application of nano-scale photo aligning (PA) layers in FLC cells. The novel photoaligned FLC devices may include field sequential color (FSC) FLC with a high resolution, high brightness, low power consumption and extended color gamut to be used for PCs, PDAs, switchable goggles, and new generation of switchable 2D/3D LCD TVs, as well as photonics elements.

  4. Construction and identification of anti-ENR phage display scFv libraries%抗恩诺沙星噬菌体单链抗体库的构建及鉴定

    Institute of Scientific and Technical Information of China (English)

    温凯; 沈建忠; 孟辉; 吴聪明; 王战辉; 张素霞

    2011-01-01

    本研究以兽药恩诺沙星为对象,构建噬菌体抗体库,为低成本、快速制备目的抗体提供新的途径.以恩诺沙星-鸡卵清蛋白(ENR-OVA)为免疫原对Balb/c小鼠进行免疫,取其脾细胞提取总RNA,并分别扩增全套抗体轻、重链基因,通过重叠延伸PCR技术,以编码柔性多肽(Gly3Ser)4的基因为接头,将轻重链基因组装为完整的scFv基因,将之克隆入pCANTAB5E载体,转化大肠杆菌XL1-Blue,以辅助噬菌体M13KO7对其进行超感染,构建噬菌体抗体库并进行富集、筛选和鉴定;构建了库容量约为2.2×106的抗恩诺沙星噬菌体单链抗体库,并筛选出26株阳性克隆,为表达单链抗体、建立免疫检测方法奠定基础.%This study focuses on construction and identification of immunized phage display library from splenocytes of hyperimmunized BALB/C mice for screening and isolation of scFv fragment against ENR, an alternative method to produce antibodies for veterinary drug residues detection. The total RNA was isolated from splenocytes of a BALB/C mouse hyperimmunized with the Enrofloxacin conjugated to chicken OVA. Variable light and heavy domains of the immunoglobulin genes were amplified by PCR and assembled to produce full-length single-chain Fv(scFV) by overlap extension PCR using a linker primer containing flexible polypeptide-(Gly3Ser)4. The scFv DNA fragment was ligated into phagemid vector pCANTAB5E and electroporated into E. coli XL1-Blue cells. The transformed cells were rescued by M13KO7 helper phage and phage libraries were constructed. The size of antibody libraries is 2.2 × 106. Following the construction of phage display scFv libraries, the recombinant phage displaying scFv were enriched and identified. There are 26 clones against ENR generated in this study.

  5. A Label-Free Electrochemical Impedance Cytosensor Based on Specific Peptide-Fused Phage Selected from Landscape Phage Library

    Science.gov (United States)

    Han, Lei; Liu, Pei; Petrenko, Valery A.; Liu, Aihua

    2016-02-01

    One of the major challenges in the design of biosensors for cancer diagnosis is to introduce a low-cost and selective probe that can recognize cancer cells. In this paper, we combined the phage display technology and electrochemical impedance spectroscopy (EIS) to develop a label-free cytosensor for the detection of cancer cells, without complicated purification of recognition elements. Fabrication steps of the cytosensing interface were monitored by EIS. Due to the high specificity of the displayed octapeptides and avidity effect of their multicopy display on the phage scaffold, good biocompatibility of recombinant phage, the fibrous nanostructure of phage, and the inherent merits of EIS technology, the proposed cytosensor demonstrated a wide linear range (2.0 × 102 ‑ 2.0 × 108 cells mL‑1), a low limit of detection (79 cells mL‑1, S/N = 3), high specificity, good inter-and intra-assay reproducibility and satisfactory storage stability. This novel cytosensor designing strategy will open a new prospect for rapid and label-free electrochemical platform for tumor diagnosis.

  6. A Label-Free Electrochemical Impedance Cytosensor Based on Specific Peptide-Fused Phage Selected from Landscape Phage Library.

    Science.gov (United States)

    Han, Lei; Liu, Pei; Petrenko, Valery A; Liu, Aihua

    2016-02-24

    One of the major challenges in the design of biosensors for cancer diagnosis is to introduce a low-cost and selective probe that can recognize cancer cells. In this paper, we combined the phage display technology and electrochemical impedance spectroscopy (EIS) to develop a label-free cytosensor for the detection of cancer cells, without complicated purification of recognition elements. Fabrication steps of the cytosensing interface were monitored by EIS. Due to the high specificity of the displayed octapeptides and avidity effect of their multicopy display on the phage scaffold, good biocompatibility of recombinant phage, the fibrous nanostructure of phage, and the inherent merits of EIS technology, the proposed cytosensor demonstrated a wide linear range (2.0 × 10(2) - 2.0 × 10(8) cells mL(-1)), a low limit of detection (79 cells mL(-1), S/N = 3), high specificity, good inter-and intra-assay reproducibility and satisfactory storage stability. This novel cytosensor designing strategy will open a new prospect for rapid and label-free electrochemical platform for tumor diagnosis.

  7. New Web Technologies for the LHCb Online Monitoring Displays

    CERN Document Server

    Lagou, Charalampia

    2017-01-01

    The LHCb Online Monitoring Displays is a web application, that gives access to real-time measurements and status information about the LHCb detector and its components, without the need to login. It is hosted at CERN on the computer lbcomet.cern.ch. The system is architecturally complex, based on the Comet technology for the data-transfer and the STOMP protocol for the communication between the clients and the message broker. The application is functional, however concerns are expressed over the future maintenance of the system’s architecture as is. The cause of these concerns are firstly the fact that the STOMP JavaScript client package is outdated and flagged by the original author flagged as non-maintained and secondly that todays modern browsers support real-time bi-directional communication which, at the time of development was not compatible even with some of the major browsers. Therefore, the objective of this project is to investigate modern data-push mechanisms, which could complement or replace...

  8. Produção e caracterização da porção Fab do anticorpo anti-digoxina utilizando a tecnologia de phage display.

    OpenAIRE

    Viviane Midori Murata

    2012-01-01

    A digoxina é um medicamento usado para tratar distúrbios cardíacos, com janela terapêutica muito estreita. Para combater seu efeito tóxico, fragmentos Fab do anticorpo policlonal anti-digoxina estão disponíveis comercialmente. Nosso objetivo foi a obtenção de variantes de fragmentos Fab do anticorpo monoclonal anti-digoxina usando a tecnologia phage display, que permite gerar fragmentos de anticorpos de alta afinidade e especificidade. Uma biblioteca combinatória de fragmentos Fab anti-digoxi...

  9. Construct breast carcinoma T7 phage display cDNA library%乳腺癌T7噬菌体展示cDNA文库的构建

    Institute of Scientific and Technical Information of China (English)

    张涛; 施宝民; 王洪; 陈鹊汀; 季堃; 余松林

    2015-01-01

    目的 构建乳腺癌组织T7噬菌体展示cDNA文库,为下一步筛选差异蛋白打下基础.方法 利用乳腺癌新鲜标本,提取总RNA,分离mRNA并进行纯化,然后合成cDNA,连接体外包装获得T7噬菌体展示cDNA文库.结果 总RNA经检测,A260/A280=1.87,纯化的mRNA产量为4.0μg,A260/A280=1.91,合成的cDNA大小在200~6 000 bp之间,原始文库的容量为2×107pfu,文库重组率为90%,插入片段长度在300~2 000 bp之间.结论 噬菌体展示技术是进行蛋白质功能研究的高效方法,构建高质量的乳腺癌噬菌体展示cDNA文库,可用于肿瘤标志物的筛选、肿瘤疫苗的研制、多肽药物的开发、靶向治疗的研究等众多领域.%Objective To construct the breast carcinoma T7 phage display cDNA library,so the foundation for the screening of differentially expressed proteins was laid. Methods Firstly,fresh specimens of breast carcinoma was used to extract total RNA,separating and purifying of mRNA. Then synthesis cDNA was done. At last,the packaging was connected in vitro and T7 phage display cDNA library was obtained. Results The total RNA was tested,the result is A260/A280=1.87. The purified mRNA is 4.0μg,A260/A280=1.91. The size of cDNA is 200-6 000 bp. The primary library capacity is 2 × 107pfu. Recombination rate of the library is 90%. The length of inserted fragments is 300-2 000 bp. Conclusions Phage display is an efficient method of protein function research. We constructed a high quality breast cancer phage display cDNA Library. The library can be used for screening tumor markers,tumor vaccine,polypeptide drug development,targeted research,and so on.

  10. Real-Time Beam Loss Monitor Display Using FPGA Technology

    CERN Document Server

    North, Matt R W

    2005-01-01

    This paper outlines the design of a Real-time Beam Loss Monitor Display for the ISIS Synchrotron based at Rutherford Appleton Laboratory (Oxon, UK). Beam loss is monitored using 39 argon filled ionisation chambers positioned around the synchrotron, the levels of which are sampled four times in each cycle. The new BLM display acquires the signals and displays four histograms, each relating to an individual sample period; the data acquisition and signal processing required to build the display fields are completed within each machine cycle (50 Hz). Attributes of the new system include setting limits for individual monitors; displaying over-limit detection, and freezing the display field when a beam trip has occurred. The design is based around a reconfigurable Field Programmable Gate Array, interfacing to a desktop monitor via the VGA standard. Results gained using simulated monitor signals have proven the system.

  11. Recombinant phage probes for Listeria monocytogenes

    Energy Technology Data Exchange (ETDEWEB)

    Carnazza, S; Gioffre, G; Felici, F; Guglielmino, S [Department of Microbiological, Genetic and Molecular Sciences, University of Messina, Messina (Italy)

    2007-10-03

    Monitoring of food and environmental samples for biological threats, such as Listeria monocytogenes, requires probes that specifically bind biological agents and ensure their immediate and efficient detection. There is a need for robust and inexpensive affinity probes as an alternative to antibodies. These probes may be recruited from random peptide libraries displayed on filamentous phage. In this study, we selected from two phage peptide libraries phage clones displaying peptides capable of specific and strong binding to the L. monocytogenes cell surface. The ability of isolated phage clones to interact specifically with L. monocytogenes was demonstrated using enzyme-linked immunosorbent assay (ELISA) and confirmed by co-precipitation assay. We also assessed the sensitivity of phage-bacteria binding by PCR on phage-captured Listeria cells, which could be detected at a concentration of 10{sup 4} cells ml{sup -1}. In addition, as proof-of-concept, we tested the possibility of immobilizing the affinity-selected phages to a putative biosensor surface. The quality of phage deposition was monitored by ELISA and fluorescent microscopy. Phage-bacterial binding was confirmed by high power optical phase contrast microscopy. Overall, the results of this work validate the concept of affinity-selected recombinant filamentous phages as probes for detecting and monitoring bacterial agents under any conditions that warrant their recognition, including in food products.

  12. Recombinant phage probes for Listeria monocytogenes

    Science.gov (United States)

    Carnazza, S.; Gioffrè, G.; Felici, F.; Guglielmino, S.

    2007-10-01

    Monitoring of food and environmental samples for biological threats, such as Listeria monocytogenes, requires probes that specifically bind biological agents and ensure their immediate and efficient detection. There is a need for robust and inexpensive affinity probes as an alternative to antibodies. These probes may be recruited from random peptide libraries displayed on filamentous phage. In this study, we selected from two phage peptide libraries phage clones displaying peptides capable of specific and strong binding to the L. monocytogenes cell surface. The ability of isolated phage clones to interact specifically with L. monocytogenes was demonstrated using enzyme-linked immunosorbent assay (ELISA) and confirmed by co-precipitation assay. We also assessed the sensitivity of phage-bacteria binding by PCR on phage-captured Listeria cells, which could be detected at a concentration of 104 cells ml-1. In addition, as proof-of-concept, we tested the possibility of immobilizing the affinity-selected phages to a putative biosensor surface. The quality of phage deposition was monitored by ELISA and fluorescent microscopy. Phage-bacterial binding was confirmed by high power optical phase contrast microscopy. Overall, the results of this work validate the concept of affinity-selected recombinant filamentous phages as probes for detecting and monitoring bacterial agents under any conditions that warrant their recognition, including in food products.

  13. Screening of the phage-display random peptide specific to the sera of patients with lung cancer%应用噬菌体随机肽库筛选肺癌血清标志物的研究

    Institute of Scientific and Technical Information of China (English)

    李文辉; 苏丽菊; 杨桐树; 杨威; 钟志; 马玉杰; 孟晓燕; 徐宁

    2012-01-01

    Objective In order to screening the phage - display random 12 peptide specific to the lung cancer and explore early diagnosis, differential diagnosis and clinical treatment for lung cancer. Methods We sdected the serum of lung cancer patient,benign tumor and normal tumor,which was 35 case respectirely,Using the phage random 12 peptide library screening^ the lung cancer specific phage clones),we pooled these serum as a screening ligand. Then, the Dot - ELISA is used to identify the lung cancer specific phage clones reactive to sera of the lung cancer patients and normal controls individually. Results Total 12 of the phage - display random 12 peptide are obtained by phage peptide library screening and the Dot - ELISA identification. Mix of positive cloning respectively with the 12 cases of lung cancer patients and 12 cases of normal serum reaction and 12 cases of lung cancer patients serum all positive, normal serum all negative, sensitivity of 100% and specificity of 100%. Conclusion Lung cancer - specific peptides were screened by phage random peptide library maybe used to diagnosis the lung cancer,and implementing early detection and early intervention has important theoretical significance and application value of lung cancer.%目的 利用噬菌体随机12肽库筛选肺癌患者血清中标志物,对探索肺癌的早期诊断、鉴别诊断、临床治疗特别是实施针对肺癌的早期干预有重要的理论意义和应用价值.方法 分别选取正常人、良性肿瘤患者及肺癌患者血清各35例,先用肺癌患者、良性肿瘤患者、正常人混合血清作为筛选配基,对噬菌体随机12肽库进行减性淘筛,获得肺癌血清特异性结合的噬菌体克隆,并用患者混合血清进行Dot-ELISA实验鉴定获得的噬菌体克隆,进而分别用肺癌患者及正常人血清各12例进一步鉴定阳性噬菌体的混合克隆,确定阳性噬菌体克隆与个体血清之间的结合情况.结果 经减性筛选后,特异结

  14. Research on Screening Peptides Specifically Targeting Laryngeal Squamous Cell Carcinoma by Phage Display Technique%用噬菌体展示技术筛选喉癌细胞靶向肽的研究

    Institute of Scientific and Technical Information of China (English)

    冯俊; 李丽; 杨洪斌; 刘世喜

    2011-01-01

    目的 筛选人源喉癌Hep-2细胞株特异结合的短肽,作为喉癌靶向治疗的载体.方法 体外培养Hep-2细胞株作为靶细胞,人正常喉黏膜上皮细胞为吸附细胞;用噬菌体展示十二肽库进行3轮差减筛选,随机挑取10个噬菌体克隆进行测序;采用酶联免疫吸附(enzyme linked immunosorbent assay,ELISA)法鉴定噬菌体与Hep-2细胞的结合活性;通过免疫荧光鉴定喉癌细胞特异性结合肽(F2)噬菌体阳性克隆与喉癌细胞结合的特异性.结果 经过3轮筛选后,噬菌体在靶细胞Hep-2上出现明显富集;ELISA分析鉴定显示5个阳性克隆能与Hep-2细胞特异结合,其中F2噬菌体克隆对喉癌细胞的结合靶向性明显高于对照细胞(P<0.05);免疫荧光显色显示,F2能特异性地与喉癌细胞结合.结论 利用噬菌体展示肽库技术,可以成功筛选到F2,其可能成为喉癌靶向治疗的载体.%Objective To obtain the polypeptides specifically bound to laryngeal squamous cell carcinoma line (Hep-2) and use it as a potential therapeutic vector targeting laryngeal squamous cell carcinoma patients. Methods With the Hep-2 cells as the target cells and human normal laryngeal squamous epithelial cells (HNLE cells) as the absorber cells, 3 rounds of panning from a Ph. D. -12TM phage-display peptide library were carried out. Ten randomly selected phage clones were sent for sequence detection. The affinity of phage clones was detected by enzyme-linked immunosorbent assay (ELISA). The positive phage clones (F2) specifically bound to Hep-2 were identified by immunofluorescence detection. Results After 3 rounds of screening, 5 positive phage clones showed specific binding to Hep-2 cells and the affinity of positive phage clones (F2) was significantly higher than that of the control groups (P<0. 05). The results of immunofluorescence detection indicated that F2 could be specifically bound to Hep-2. Conclusion Phage display peptide libraries technique can

  15. Phage displaying peptides mimic schistosoma antigenic epitopes selected by rat natural antibodies and protective immunity induced by their immunization in mice

    Institute of Scientific and Technical Information of China (English)

    Min Wang; Xin-Yuan Yi; Xian-Ping Li; Dong-Ming Zhou; McReynolds Larry; Xian-Fang Zeng

    2005-01-01

    AIM: To obtain the short peptides mimic antigenic epitopes selected by rat natural antibodies to schistosomes, and to explore their immunoprotection against schistosomiasis in mice.METHODS: Adults worm antigens (AWA) were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and enzyme-linked transferred immunoblotting methods with normal SD rat sera (NRS). The killing effects on schistosomula with fresh and heat-inactivated sera from SD rats were observed. Then the purified IgG from sera of SD rats was used to biopan a phage random peptide library and 20 randomly selected positive clones were detected by ELISA and 2 of them were sequenced.Sixty female mice were immunized thrice with positive phage clones (0, 2nd, 4th wk). Each mouse was challenged with 40 cercariae, and all mice were killed 42 d after challenge. The worms and the liver eggs were counted. RESULTS: NRS could specifically react to the molecules of 75 000, 47 000, 34 500 and 23 000 of AWA. Sera from SD rats showed that the mortality rate of schistosomula was 76.2%, and when the sera were heat-inactivated in vitro, the mortality rate was decreased to 41.0% after being cultured for 48 h. The specific phages bound to IgG were enriched about 300-folds after three rounds of biopanning. Twenty clones were detected by ELISA, 19 of them bound to the specific IgG of rat sera. Immunization with these epitopes was carried out in mice. Compared with the control groups, the mixture of two mimic peptides could induce 34.9% (P = 0.000) worm reduction and 67.6% (P = 0.000) total liver egg reduction in mice. Two different mimic peptides could respectively induce 31.0% (P = 0.001), 14.5% (P = 0.074) worm reduction and 61.2% (P = 0.000), 35.7% (P = 0.000) total liver egg reduction. The specific antibody could be induced by immunization of the mimic peptides, and the antibody titer in immunized mice reached more than 1:6 400 as detected by ELISA.CONCLUSION: Specific peptides mimic antigenic

  16. Application of whole-cell subtractive panning in phage display library screening%全细胞差减筛选法在噬菌体展示文库筛选中的应用

    Institute of Scientific and Technical Information of China (English)

    吴红珍; 张林波

    2012-01-01

    全细胞差减筛选法是近年来在细胞筛选的基础上发展起来的一项筛选技术,其利用成对的细胞,即两种不同状态的细胞对噬菌体展示文库进行差减筛选,主要用于筛选新的抗原表位、受体、配体、新型疫苗、肿瘤细胞的靶向活性肽、靶向基因载体及受体或酶的激动剂或抑制剂等.本文就其筛选的原理、优缺点、优化及其在噬菌体展示文库筛选中的应用作一综述.%Whole-cell subtractive panning is a screening technique developed based on whole-cell screening in recent years, which performs subtractive panning on phage display library by using couple cells, I.e. Two states of cells. It is mainly used for screening of novel antigenic epitopes, receptors, ligands, novel vaccines, the targeted active peptides of tumor cells, targeted gene vectors, and stimulants or antagonists of receptors or enzyme etc. The principle, advantages, disadvantages and optimization of the technique as well as its application in phage display library screening are reviewed in this paper.

  17. Assembly of the small outer capsid protein, Soc, on bacteriophage T4: a novel system for high density display of multiple large anthrax toxins and foreign proteins on phage capsid.

    Science.gov (United States)

    Li, Qin; Shivachandra, Sathish B; Zhang, Zhihong; Rao, Venigalla B

    2007-07-27

    Bacteriophage T4 capsid is a prolate icosahedron composed of the major capsid protein gp23*, the vertex protein gp24*, and the portal protein gp20. Assembled on its surface are 810 molecules of the non-essential small outer capsid protein, Soc (10 kDa), and 155 molecules of the highly antigenic outer capsid protein, Hoc (39 kDa). In this study Soc, a "triplex" protein that stabilizes T4 capsid, is targeted for molecular engineering of T4 particle surface. Using a defined in vitro assembly system, anthrax toxins, protective antigen, lethal factor and their domains, fused to Soc were efficiently displayed on the capsid. Both the N and C termini of the 80 amino acid Soc polypeptide can be simultaneously used to display antigens. Proteins as large as 93 kDa can be stably anchored on the capsid through Soc-capsid interactions. Using both Soc and Hoc, up to 1662 anthrax toxin molecules are assembled on the phage T4 capsid under controlled conditions. We infer from the binding data that a relatively high affinity capsid binding site is located in the middle of the rod-shaped Soc, with the N and C termini facing the 2- and 3-fold symmetry axes of the capsid, respectively. Soc subunits interact at these interfaces, gluing the adjacent capsid protein hexamers and generating a cage-like outer scaffold. Antigen fusion does interfere with the inter-subunit interactions, but these interactions are not essential for capsid binding and antigen display. These features make the T4-Soc platform the most robust phage display system reported to date. The study offers insights into the architectural design of bacteriophage T4 virion, one of the most stable viruses known, and how its capsid surface can be engineered for novel applications in basic molecular biology and biotechnology.

  18. Synthetic Phage for Tissue Regeneration

    Directory of Open Access Journals (Sweden)

    So Young Yoo

    2014-01-01

    Full Text Available Controlling structural organization and signaling motif display is of great importance to design the functional tissue regenerating materials. Synthetic phage, genetically engineered M13 bacteriophage has been recently introduced as novel tissue regeneration materials to display a high density of cell-signaling peptides on their major coat proteins for tissue regeneration purposes. Structural advantages of their long-rod shape and monodispersity can be taken together to construct nanofibrous scaffolds which support cell proliferation and differentiation as well as direct orientation of their growth in two or three dimensions. This review demonstrated how functional synthetic phage is designed and subsequently utilized for tissue regeneration that offers potential cell therapy.

  19. Construction and screening of phage display single chain antibody library against histidine-rich protein Ⅱ of Plasmodium falciparum%抗恶性疟原虫富含组氨酸蛋白Ⅱ单链抗体库的构建及筛选

    Institute of Scientific and Technical Information of China (English)

    侯云霞; 董文其; 徐伟文; 王萍; 陈白虹; 李明

    2001-01-01

    Objective To construct phage display single-chain antibody fragments (scFvs) library against histidine-rich protein Ⅱ (HRP-Ⅱ) of Plasmodium falciparum and select specific scFvs of anti- HRP-Ⅱ for the purpose of malaria diagnosis. Method The genes of variable fragments of heavy chain (VH) and light chain (VL) were gained from the spleen cells of BALB/c mice immunized with HRP-Ⅱ protein. The VH and VL genes were then assembled by the method of splicing overlapping extension and cloned into phagemid vector pCANTAB 5E. The scFv phage antibodies were expressed at the surface of the phage after the rescue by helper phage M13K07. HRP- Ⅱ protein was used as antigenic reagent for panning and screening. Results The total RNA from the spleen cells was isolated, and cDNA obtained and VH and VL gene regions amplified using PCR. The VH and VL gene regions were combined with a flexible linker ligated into the pCANTAB 5E phagemid vector, and transformed into TG1 Escherichia coli. The repertoire of the phage antibody was about 106. After panning and screening, 8 positive clones expressed scFv antibodies which were specific for HPR-Ⅱ as demonstrated by ELISA. Conclusion Phage display technology can be used as a powerful tool in making scFv antibodies which have the potential to be used as reagents in the diagnosis and therapy of malaria.%目的构建抗恶性疟原虫富含组氨酸蛋白Ⅱ(Histidine- rich protein II, HRP-II)单链抗体库,并筛选出阳性克隆。方法用噬菌体抗体库技术构建抗恶性疟原虫HRP-Ⅱ单链抗体库,并以HRP-Ⅱ为靶抗原对该库进行了三轮"亲和吸附-援救-感染扩增"的富集,挑取单菌落筛选并鉴定阳性克隆。结果获得目的基因并成功构建抗恶性疟原虫HRP-Ⅱ的单链抗体库,库容为106,并从中筛选出8株阳性克隆。结论噬菌体抗体库技术具有高效的筛选性能,抗 HRP-Ⅱ单链抗体的制备为其在恶性疟的免疫快速诊断方法中的应用奠定了基础。

  20. Comparative Evaluation of Display Technologies for Collaborative Design Review

    Science.gov (United States)

    2009-04-01

    Wickens, & Chudy, 2000), in battlefield displays (Wickens, Thomas, Merlo , & Hah, 1999), and in data visualization (McCormick et al., 1998). Select ing...human performance (3rd ed.). Upper Saddle River, NJ: Prentice Hall. Wickens, C. D., Thomas, L., Merlo , J., & Hah, S. (1999). Immersion and battlefield

  1. Gold nanoprobe functionalized with specific fusion protein selection from phage display and its application in rapid, selective and sensitive colorimetric biosensing of Staphylococcus aureus.

    Science.gov (United States)

    Liu, Pei; Han, Lei; Wang, Fei; Petrenko, Valery A; Liu, Aihua

    2016-08-15

    Staphylococcus aureus (S. aureus) is one of the most ubiquitous pathogens in public healthcare worldwide. It holds great insterest in establishing robust analytical method for S. aureus. Herein, we report a S. aureus-specific recognition element, isolated from phage monoclone GQTTLTTS, which was selected from f8/8 landscape phage library against S. aureus in a high-throughput way. By functionalizing cysteamine (CS)-stabilized gold nanoparticles (CS-AuNPs) with S. aureus-specific pVIII fusion protein (fusion-pVIII), a bifunctional nanoprobe (CS-AuNPs@fusion-pVIII) for S. aureus was developed. In this strategy, the CS-AuNPs@fusion-pVIII could be induced to aggregate quickly in the presence of target S. aureus, resulting in a rapid colorimetric response of gold nanoparticles. More importantly, the as-designed probe exhibited excellent selectivity over other bacteria. Thus, the CS-AuNPs@fusion-pVIII could be used as the indicator of target S. aureus. This assay can detect as low as 19CFUmL(-1)S. aureus within 30min. Further, this approach can be applicable to detect S. aureus in real water samples. Due to its sensitivity, specificity and rapidness, this proposed method is promising for on-site testing of S. aureus without using any costly instruments.

  2. Generation of a phage-display library of single-domain camelid VH H antibodies directed against Chlamydomonas reinhardtii antigens, and characterization of VH Hs binding cell-surface antigens.

    Science.gov (United States)

    Jiang, Wenzhi; Rosenberg, Julian N; Wauchope, Akelia D; Tremblay, Jacqueline M; Shoemaker, Charles B; Weeks, Donald P; Oyler, George A

    2013-11-01

    Single-domain antibodies (sdAbs) are powerful tools for the detection, quantification, purification and subcellular localization of proteins of interest in biological research. We have generated camelid (Lama pacos) heavy chain-only variable VH domain (VH H) libraries against antigens in total cell lysates from Chlamydomonas reinhardtii. The sdAbs in the sera from immunized animals and VH H antibody domains isolated from the library show specificity to C. reinhardtii and lack of reactivity to antigens from four other algae: Chlorella variabilis, Coccomyxa subellipsoidea, Nannochloropsis oceanica and Thalassiosira pseudonana. Antibodies were produced against a diverse representation of antigens as evidenced by sera ELISA and protein-blot analyses. A phage-display library consisting of the VH H region contained at least 10(6) individual transformants, and thus should represent a wide range of C. reinhardtii antigens. The utility of the phage library was demonstrated by using live C. reinhardtii cells to pan for VH H clones with specific recognition of cell-surface epitopes. The lead candidate VH H clones (designated B11 and H10) bound to C. reinhardtii with EC50 values ≤ 0.5 nm. Treatment of cells with VH H B11 fused to the mCherry or green fluorescent proteins allowed brilliant and specific staining of the C. reinhardtii cell wall and analysis of cell-wall genesis during cell division. Such high-complexity VH H antibody libraries for algae will be valuable tools for algal researchers and biotechnologists.

  3. Isolation of ScFv antibodies of rP27Kip1 from phage display libraries constructed from immunized and non-immunized repertoires

    Institute of Scientific and Technical Information of China (English)

    曹跃琼; 乔守怡; 袁有忠; 黄建生; 赵寿元

    1999-01-01

    Through mRNA extract, RT and a series of PCR, phage antibody libraries were constructed from rP27Kiplimmunized and non-immunized mice. After only one round of selection with rP27Kipl, clones from each library were chosen randomly and digested by Taq I and Hinf I. 11 of 64 clones from the immunized animal had consistent restriction pattern, while none of the 64 clones from the non-immunized animal had, except that one had the same fragments pattern as that of the 11 clones. The 12 fragments were expressed in E. coli BL21(DE3)/pET-28b(+) system. ELISA showed that some of the fragments could bind to rP27Kipl specifically. All these results implied that specific antibody can be obtained by genetic engineering without hybridoma or many rounds of growth and panning selection.

  4. Recognition of Vipera ammodytes meridionalis neurotoxin vipoxin and its components using phage-displayed scFv and polyclonal antivenom sera.

    Science.gov (United States)

    Stoyanova, Vishnya; Aleksandrov, Radoslav; Lukarska, Maria; Duhalov, Deyan; Atanasov, Vasil; Petrova, Svetla

    2012-10-01

    Vipoxin is a potent postsynaptic heterodimeric neurotoxin isolated from the venom of the Bulgarian snake Vipera ammodytes meridionalis, whose snakebites cause different and strongly manifested pathophysiological effects (neurotoxic, hemolytic, anticoagulant, convulsant, hypotensive, hyperglycemic etc.). The neutralization of snake toxins calls for extensive research through the application of different approaches: antibodies, non-immunologic inhibitors, natural products derived from plants and animals, as well as synthetic drugs. In this study, we applied naive Tomlinson I + J (Cambridge, UK) libraries to obtain recombinant human scFv antibodies against the vipoxin's two subunits--basic and toxic phospholipase A₂ (PLA₂) and acidic, non-toxic component. We found that 33 of more than hundred tested clones were positive and recognized vipoxin and its subunits. Enriched scFv-phage samples (1.2 × 10⁹ pfu/ml) were analyzed for their binding (ELISA) and enzyme-inhibiting abilities. Single chain Fv-phage clones--D₁₂, E₃, F₆, D₁₀ and G₅ exhihest binding affinity for the toxic component. Clones A₁, D₁₂ and C₁₂ recognized preferentially vipoxin's acidic component. Clones E₃, G₅ and H₄ inhibited the enzymatic activity of both vipoxin and its purified and separated toxic subunit to the highest extent. Six of the selected clones (E₃, G₅, H₄, C₁₂, D₁₀ and A₁₁) inhibited direct hemolytic activity of vipoxin and its pure PLA₂ subunit. The obtained specific scFv antibodies will be used for epitope mapping studies required to shed light on the role of the phospholipase A₂ activity for the vipoxin toxicity and its effective neutralization. Copyright © 2012 Elsevier Ltd. All rights reserved.

  5. Flexible Display and Integrated Communication Devices (FDICD) Technology. Volume 2

    Science.gov (United States)

    2008-06-01

    David Huffman, Keith Tognoni, Robert Anderson, Michael Hack , Anna Chwang, Richard Hewitt, Robert A. Street, Jackson Ho, JengPing Lu, and Darrel G...Anderson, Michael Hack , Anna Chwang, Richard Hewitt, Robert A. Street, Jackson Ho, JengPing Lu, and Darrel G. Hopper, “Flexible Display and...fabric covering; tethered stylus; Bluetooth, WiFi 802.11b&g; Windows Mobile 5.0; and touchscreen. Ten (10) units of this spiral (Spiral 3.0) will be

  6. Phage therapy pharmacology: calculating phage dosing.

    Science.gov (United States)

    Abedon, Stephen

    2011-01-01

    Phage therapy, which can be described as a phage-mediated biocontrol of bacteria (or, simply, biocontrol), is the application of bacterial viruses-also bacteriophages or phages-to reduce densities of nuisance or pathogenic bacteria. Predictive calculations for phage therapy dosing should be useful toward rational development of therapeutic as well as biocontrol products. Here, I consider the theoretical basis of a number of concepts relevant to phage dosing for phage therapy including minimum inhibitory concentration (but also "inundation threshold"), minimum bactericidal concentration (but also "clearance threshold"), decimal reduction time (D value), time until bacterial eradication, threshold bacterial density necessary to support phage population growth ("proliferation threshold"), and bacterial density supporting half-maximal phage population growth rates (K(B)). I also address the concepts of phage killing titers, multiplicity of infection, and phage peak densities. Though many of the presented ideas are not unique to this chapter, I nonetheless provide variations on derivations and resulting formulae, plus as appropriate discuss relative importance. The overriding goal is to present a variety of calculations that are useful toward phage therapy dosing so that they may be found in one location and presented in a manner that allows facile appreciation, comparison, and implementation. The importance of phage density as a key determinant of the phage potential to eradicate bacterial targets is stressed throughout the chapter.

  7. Hepatitis A virus mimotope mapping by phage display peptide library%用噬菌体展示肽库技术筛选甲肝病毒抗原模拟表位

    Institute of Scientific and Technical Information of China (English)

    曹经瑗; 李建东; 郑惠惠; 毕胜利; 李德新

    2012-01-01

    Objective A 12 mer phage display peptide library was used to identify hepatitis A virus mimotopes of antigenic determinants,to provide the feasibility of virus epitope mapping by using this approach.Methods Using purified anti-hepatitis A virus monoclonal antibody as affinity selective molecule,phage display peptide library was biopanned and positive clones were selected by ELISA,competition assay and DNA sequencing.Results 10 ELISA positive clones were chosen for DNA sequencing,and the displayed peptide sequences were deduced.9 of them showed identical nucleotide sequence,and similarity in their amino acid sequence with VP1 of HAV HM175 was found,but no sequence homology was found between the other phage clone and the capsid proteins of HAV.Those peptides may behave as mimotopes of HAV.Conclusion The mimotope of HAV was selected by using phage display peptide library screening.The results provide the potential of this method to search for the mimotopes of the virus.%目的 用噬菌体展示肽库技术筛选甲型肝炎(甲肝)病毒抗原模拟表位,为病毒抗原决定簇定位探索可行方法.方法 用纯化的抗甲肝病毒单克隆抗体,对噬菌体展示12肽库进行3轮“吸附-洗脱-扩增”筛选,随机挑取10个克隆,用酶联免疫吸附法(ELISA)对噬菌体克隆进行抗原性鉴定、竞争抑制鉴定及DNA序列测定分析,推导出展示肽氨基酸序列并与甲肝病毒(HAV)代表株结构蛋白氨基酸序列比较.结果 10个噬菌体克隆ELISA检测全为阳性,9个具有一致序列,与HAVHM175株结构蛋白中和活性表位之一:VP1 157-171区具有类似序列,另一株噬菌体克隆在HAVHM175中未发现类似序列,结果表明这些展示肽可能是HAV抗原模拟表位.结论 用噬菌体展示肽库技术筛选得到了HAV模拟表位,为开展病毒模拟表位研究打下了基础.

  8. Use of display technologies for augmented reality enhancement

    Science.gov (United States)

    Harding, Kevin

    2016-06-01

    Augmented reality (AR) is seen as an important tool for the future of user interfaces as well as training applications. An important application area for AR is expected to be in the digitization of training and worker instructions used in the Brilliant Factory environment. The transition of work instructions methods from printed pages in a book or taped to a machine to virtual simulations is a long step with many challenges along the way. A variety of augmented reality tools are being explored today for industrial applications that range from simple programmable projections in the work space to 3D displays and head mounted gear. This paper will review where some of these tool are today and some of the pros and cons being considered for the future worker environment.

  9. The Use of Haptic Display Technology in Education

    Directory of Open Access Journals (Sweden)

    Woodrow Barfield

    2009-01-01

    Full Text Available The experience of "virtual reality" can consist of head-tracked and stereoscopic virtual worlds,spatialized sound, haptic feedback, and to a lesser extent olfactory cues. Although virtual realitysystems have been proposed for numerous applications, the field of education is one particularapplication that seems well-suited for virtual reality technology. Recently, in the field ofeducation, virtual reality systems equipped with haptic devices, have been used to assist in thelearning process. The main benefit of haptic technology for education is that it increases therealism of simulations by providing force or tactile feedback to the user. Such feedback can beused to model the atomic orbits of electrons, feel of tissue of the abdomen during a laparoscopictraining exercise, or visualize and interact with nanoscale materials. This article discussesthe basics of the haptic sense, discusses a few common haptic devices, and concludeswith current applications of haptics in education.

  10. Floating image device with autostereoscopic display and viewer-tracking technology

    Science.gov (United States)

    Chen, Chang-Ying; Tseng, Kun-Lung; Wang, Chy-Lin; Tsai, Chao-Hsu

    2012-03-01

    Now, numerous types of 3D display have been developed or under-developing. However, most of them present stereoscopic images in a space with limited distance from the physical screen. A display which can deliver stereoscopic images in the free space and satisfies the touching sense of viewers is always expected. As a result, we proposed a floating image device with auto-stereoscopic display and viewer tracking technology. The key technology includes that the optical projected lenses with wide view angle which exceeds 30 degrees, the optimized parameters of 2 views auto-stereoscopic display which fits the viewing specifications and the viewer tracking technology which can update the corresponding image of the particular view angle in real time. The novel display is the other choice for consumers especially for product exhibition, user interface of kiosk and a kind of apparatus of video conference, etc.

  11. Designing phage therapeutics.

    Science.gov (United States)

    Goodridge, Lawrence D

    2010-01-01

    Phage therapy is the application of phages to bodies, substances, or environments to effect the biocontrol of pathogenic or nuisance bacteria. To be effective, phages, minimally, must be capable of attaching to bacteria (adsorption), killing those bacteria (usually associated with phage infection), and otherwise surviving (resisting decay) until they achieve attachment and subsequent killing. While a strength of phage therapy is that phages that possess appropriate properties can be chosen from a large diversity of naturally occurring phages, a more rational approach to phage therapy also can include post-isolation manipulation of phages genetically, phenotypically, or in terms of combining different products into a single formulation. Genetic manipulation, especially in these modern times, can involve genetic engineering, though a more traditional approach involves the selection of spontaneously occurring phage mutants during serial transfer protocols. While genetic modification typically is done to give rise to phenotypic changes in phages, phage phenotype alone can also be modified in vitro, prior to phage application for therapeutic purposes, as for the sake of improving phage lethality (such as by linking phage virions to antibacterial chemicals such as chloramphenicol) or survival capabilities (e.g., via virion PEGylation). Finally, phages, both naturally occurring isolates or otherwise modified constructs, can be combined into cocktails which provide collectively enhanced capabilities such as expanded overall host range. Generally these strategies represent different routes towards improving phage therapy formulations and thereby efficacy through informed design.

  12. A decade of yeast surface display technology: where are we now?

    Science.gov (United States)

    Pepper, Lauren R; Cho, Yong Ku; Boder, Eric T; Shusta, Eric V

    2008-02-01

    Yeast surface display has become an increasingly popular tool for protein engineering and library screening applications. Recent advances have greatly expanded the capability of yeast surface display, and are highlighted by cell-based selections, epitope mapping, cDNA library screening, and cell adhesion engineering. In this review, we discuss the state-of-the-art yeast display methodologies and the rapidly expanding set of applications afforded by this technology.

  13. Evaluation of display technologies for Internet of Things (IoT)

    Science.gov (United States)

    Sabo, Julia; Fegert, Tobias; Cisowski, Matthäus Stephanus; Marsal, Anatolij; Eichberger, Domenik; Blankenbach, Karlheinz

    2017-02-01

    Internet of Things (IoT) is a booming industry. We investigated several (semi-) professional IoT devices in combination with displays (focus on reflective technologies) and LEDs. First, these displays were compared for reflectance and ambient light performance. Two measurement set-ups with diffuse conditions were used for simulating typical indoor lighting conditions of IoT displays. E-paper displays were evaluated best as they combine a relative high reflectance with large contrast ratio. Reflective monochrome LCDs show a lower reflectance but are widely available. Second we studied IoT microprocessors interfaces to displays. A µP can drive single LEDs and one or two Seg 8 LED digits directly by GPIOs. Other display technologies require display controllers with a parallel or serial interface to the microprocessor as they need dedicated waveforms for driving the pixels. Most suitable are display modules with built-in display RAM as only pixel data have to be transferred which changes. A HDMI output (e.g. Raspberry Pi) results in high cost for the displays, therefore AMLCDs are not suitable for low to medium cost IoT systems. We compared and evaluated furthermore status indicators, icons, text and graphics IoT display systems regarding human machine interface (HMI) characteristics and effectiveness as well as power consumption. We found out that low resolution graphics bistable e-paper displays are the most appropriate display technology for IoT systems as they show as well information after a power failure or power switch off during maintenance or e.g. QR codes for installation. LED indicators are the most cost effective approach which has however very limited HMI capabilities.

  14. Three-Dimensional Display Technologies for Anatomical Education: A Literature Review

    Science.gov (United States)

    Hackett, Matthew; Proctor, Michael

    2016-08-01

    Anatomy is a foundational component of biological sciences and medical education and is important for a variety of clinical tasks. To augment current curriculum and improve students' spatial knowledge of anatomy, many educators, anatomists, and researchers use three-dimensional (3D) visualization technologies. This article reviews 3D display technologies and their associated assessments for anatomical education. In the first segment, the review covers the general function of displays employing 3D techniques. The second segment of the review highlights the use and assessment of 3D technology in anatomical education, focusing on factors such as knowledge gains, student perceptions, and cognitive load. The review found 32 articles on the use of 3D displays in anatomical education and another 38 articles on the assessment of 3D displays. The review shows that the majority (74 %) of studies indicate that the use of 3D is beneficial for many tasks in anatomical education, and that student perceptions are positive toward the technology.

  15. Geowall: Investigations into Low-Cost Stereo Display Technologies

    Science.gov (United States)

    Steinwand, Daniel R.; Davis, Brian; Weeks, Nathan

    2003-01-01

    Recently, the combination of new projection technology, fast, low-cost graphics cards, and Linux-powered personal computers has made it possible to provide a stereoprojection and stereoviewing system that is much more affordable than previous commercial solutions. These Geowall systems are low-cost visualization systems built with commodity off-the-shelf components, run on open-source (and other) operating systems, and using open-source applications software. In short, they are ?Beowulf-class? visualization systems that provide a cost-effective way for the U. S. Geological Survey to broaden participation in the visualization community and view stereoimagery and three-dimensional models2.

  16. Competing Technologies and Industry Evolution: The Benefits of Making Mistakes in the Flat Panel Display Industry

    OpenAIRE

    Eggers, J.P.

    2010-01-01

    Managers at firms facing uncertain competing technologies evolving concurrently face a complex decision set, including options to invest in one technology or other, both technologies, or to wait to invest. This study investigates the role that experience, learning and timing play in affecting the firm-level pros and cons of each of these four strategies in a technological competition situation. Using a unique data set on the evolution of the global flat panel display industry, this study offe...

  17. Feasibility Study of Integrating IDELIX’s Pliable Display Technology into the COPlanS Technology Demonstration Software

    Science.gov (United States)

    2005-03-31

    Feasibility Study of Integrating IDELIX’s Pliable Display Technology into the COPlanS Technology Demonstration Software Issue Number: Version 1... Technology Demonstration Software 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER 5e. TASK NUMBER 5f...Collaborative Operations Planning System (COPlanS) technology demonstration software in the areas of collaboration and data visualization using IDELIX’s Pliable

  18. Three-dimensional display technologies of recent interest: principles, status, and issues [Invited].

    Science.gov (United States)

    Hong, Jisoo; Kim, Youngmin; Choi, Hee-Jin; Hahn, Joonku; Park, Jae-Hyeung; Kim, Hwi; Min, Sung-Wook; Chen, Ni; Lee, Byoungho

    2011-12-01

    Recent trends in three-dimensional (3D) display technologies are very interesting in that both old-fashioned and up-to-date technologies are being actively investigated together. The release of the first commercially successful 3D display product raised new research topics in stereoscopic display. Autostereoscopic display renders a ray field of a 3D image, whereas holography replicates a wave field of it. Many investigations have been conducted on the next candidates for commercial products to resolve existing limitations. Up-to-date see-through 3D display is a concept close to the ultimate goal of presenting seamless virtual images. Although it is still far from practical use, many efforts have been made to resolve issues such as occlusion problems.

  19. Anti-aliasing in Aircraft Cockpit Display System Based on Modified Bresenham Algorithm and Virtual Technology

    Directory of Open Access Journals (Sweden)

    Dan Sun

    2014-06-01

    Full Text Available In this paper, an improved Bresenham algorithm is proposed in order to improve the display effect of the digital instrument display systems in aircraft and aviation simulators with following the ARINC 661 specification. According to the algorithm, the pixel brightness is calculated according to the proportional relation of the distance to the pixel for realizing the anti-aliasing. In Combine with areal sampled and double buffer image processing technology, the idea can increase the operation efficiency compared with the traditional method. In accordance with the analysis of the ARINC 661, the air data system instrument is implemented in the VAPS. Experimental results reveal that the improved algorithm and digital image processing technology can indeed solve display distortion problems more effectively and accurately, the display effect is improved obviously. The implemented schemes can achieve the airborne electronic display system on the high performance and satisfy aircraft airworthiness requirements and standards

  20. Display MTF measurements based on scanning and imaging technologies and its importance in the application space

    Science.gov (United States)

    Kaur, Balvinder; Olson, Jeff; Flug, Eric A.

    2016-05-01

    Measuring the Modulation Transfer Function (MTF) of a display monitor is necessary for many applications such as: modeling end-to-end systems, conducting perception experiments, and performing targeting tasks in real-word scenarios. The MTF of a display defines the resolution properties and quantifies how well the spatial frequencies are displayed on a monitor. Many researchers have developed methods to measure display MTFs using either scanning or imaging devices. In this paper, we first present methods to measure display MTFs using two separate technologies and then discuss the impact of a display MTF on a system's performance. The two measurement technologies were scanning with a photometer and imaging with a CMOS based camera. To estimate a true display MTF, measurements made with the photometer were backed out for the scanning optics aperture. The developed methods were applied to measure MTFs of the two types of monitors, Cathode Ray Tube (CRT) and Liquid Crystal Display (LCD). The accuracy of the measured MTFs was validated by comparing MTFs measured with the two systems. The methods presented here are simple and can be easily implemented employing either a Prichard photometer or an imaging device. In addition, the impact of a display MTF on the end-to-end performance of a system was modeled using NV-IPM.

  1. Modulation of the CD40-CD40 ligand interaction using human anti-CD40 single-chain antibody fragments obtained from the n-CoDeR phage display library.

    Science.gov (United States)

    Ellmark, Peter; Ottosson, Camilla; Borrebaeck, Carl A K; Malmborg Hager, Ann-Christin; Furebring, Christina

    2002-08-01

    CD40 plays a central regulatory role in the immune system and antibodies able to modulate CD40 signalling may consequently have a potential in immunotherapy, in particular for treatment of lymphomas and autoimmune disease like multiple sclerosis. As a first step to achieve this goal, we describe the selection and characterization of a novel set of fully human anti-CD40 antibody fragments (scFv) from a phage display library (n-CoDeR). In order to determine their biological potential, these antibody fragments have been analysed for their ability to promote B-cell activation, rescue from apoptosis and to block the CD40-CD40 ligand (CD40L) interaction. The selected cohort of human scFv could be subcategorized, each expressing a distinct functional signature. Thus scFv were generated that induced B-cell proliferation, rescued B cells from apoptosis and blocked the CD40-CD40L interaction to different extents. In particular, one of the scFv clones (F33) had the ability to abrogate completely this interaction. The epitope recognition patterns as well as individual rate constants were also determined and the affinity was shown to vary from low to high nanomolar range. In conclusion, this panel of human anti-CD40 scFv fragments displays a number of distinct properties, which may constitute a valuable source when evaluating candidates for in vivo trials.

  2. Tutorial on the Psychophysics and Technology of Virtual Acoustic Displays

    Science.gov (United States)

    Wenzel, Elizabeth M.; Null, Cynthia (Technical Monitor)

    1998-01-01

    the localization of real and synthesized stimuli are directly compared in psychophysical studies. To this end, the results of psychophysical experiments examining the perceptual validity of the synthesis technique will be reviewed and factors that can enhance perceptual accuracy and realism will be discussed. Of particular interest is the relationship between individual differences in HRTFs and in behavior, the role of reverberant cues in reducing the perceptual errors observed with virtual sound sources, and the importance of developing perceptually valid methods of simplifying the synthesis technique. Recent attempts to implement the synthesis technique in real time systems will also be discussed and an attempt made to interpret their quoted system specifications in terms of perceptual performance. Finally, some critical research and technology development issues for the future will be outlined.

  3. Driving technology for improving motion quality of active-matrix organic light-emitting diode display

    Science.gov (United States)

    Kim, Jongbin; Kim, Minkoo; Kim, Jong-Man; Kim, Seung-Ryeol; Lee, Seung-Woo

    2014-09-01

    This paper reports transient response characteristics of active-matrix organic light emitting diode (AMOLED) displays for mobile applications. This work reports that the rising responses look like saw-tooth waveform and are not always faster than those of liquid crystal displays. Thus, a driving technology is proposed to improve the rising transient responses of AMOLED based on the overdrive (OD) technology. We modified the OD technology by combining it with a dithering method because the conventional OD method cannot successfully enhance all the rising responses. Our method can improve all the transitions of AMOLED without modifying the conventional gamma architecture of drivers. A new artifact is found when OD is applied to certain transitions. We propose an optimum OD selection method to mitigate the artifact. The implementation results show the proposed technology can successfully improve motion quality of scrolling texts as well as moving pictures in AMOLED displays.

  4. Magnetic Affinity Immunoassay Based Enzyme-Labeled Phage Displayed Antibody%基于酶标噬菌体抗体的磁分离免疫分析方法

    Institute of Scientific and Technical Information of China (English)

    穆晞惠; 童朝阳; 黄启斌; 刘冰; 刘志伟; 郝兰群; 张金平

    2014-01-01

    A new magnetic affinity immunoassay (MAIA) strategy based on enzyme-labeled phage displayed antibody was developed. The assay consisted of a sandwich format in which immobilized polyclonal antibody (pcAb) on magnetic microparticle was used for capture probe, and enzyme-labeled phage displayed antibody for specific detection probe to increase enzyme amount and enhance detection signal. By the proposed method,β-bungarotoxin (β-BGT) was successfully detected. A linear relationship between absorbance value and the concentration of β-BGT in the range of 0. 016-62. 5 μg / L was obtained. The linear regression equation was Y=0. 641X+1. 355 (R =0. 9925, n = 13, p<0. 0001) with a detection limit of 0. 016 μg / L. In comparison with the traditional ELISA, this method gave a 10-fold better sensitivity in β-BGT detection. This strategy also gave a 4-fold better sensitivity comparing with the MAIA based on enzyme labeled monoclonal antibody (mcAb). Due to low detection limit, acceptable reproducibility and high specificity, this method holds great promise in toxin trace detection.%以磁微粒偶联多抗为磁性捕获探针,酶标噬菌体抗体为特异信号检测探针,采用“磁性捕获探针-待测物-酶标噬菌体抗体探针”的检测模式,成功建立了一种基于酶标噬菌体抗体的磁分离免疫分析方法。本方法检测β-银环蛇毒素线性范围为0.016~62.5μg/ L,回归方程为 Y =0.641X+1.355( R =0.9925,n =13, p<0.0001),检出限为0.016μg/ L。本方法比传统 ELISA 法检测灵敏度提高了10倍,与采用酶标单抗复合物探针的双抗体夹心磁分离免疫分析法相比,检测灵敏度提高4倍。本方法灵敏度高,具有较好重现性与特异性,在毒素的痕量检测方面具有广阔的应用前景。

  5. Using phage display to map the binding epitope of the Bacillus thuringiensis Cry2Ab toxin%利用噬菌体展示技术筛选Cry2Ab毒素受体表位的方法

    Institute of Scientific and Technical Information of China (English)

    齐佳; 刘晨曦; 吴孔明

    2012-01-01

    苏云金芽孢杆菌(Bacillus thuringiensis,Bt)产生的内毒素具有杀虫活性,Cry2Ab毒素作为Bt棉花的杀虫活性蛋白,其在靶标昆虫体内的结合受体及作用位点尚不清楚,本研究采用噬菌体展示( phage display)的方法,经4轮的“吸附-洗脱-扩繁”筛选,并对阳性克隆所携带的外源DNA片段进行序列测定后,得到2段能够与活化Cry2Ab毒素相互作用的多肽序列,通过酶联免疫结合试验(ELISA)进一步证明,这2段多肽序列与活化Cry2Ab毒素具有较高的亲和力和特异性,结果表明,利用该方法能够由噬菌体随机肽库中高效捕获亲和序列,筛选到与活化Cry2Ab毒素具有高亲和力的多肽,该序列可以模拟Cry2Ab毒素的受体表位,为进一步研究Cry2Ab毒素作用机制奠定了基础,并为今后田间抗性基因频率检测,以及毒素-受体作用机制研究工作提供更有力的技术支持.%The insecticidal Cry toxins produced by Bacillus thuringiensis are highly specific to different insects. Various proteins, such as cadherin, aminopeptidase-N ( APN) and alkaline phosphatase ( ALP) are characterized as potential Cry-receptors. However, little is known about the mode of Cry2Ab action, such as its receptors and binding sites. We used phage display to characterize the binding epitope of the Cry2Ab toxin in vitro. A two peptide sequence was identified after four-rounds of screening. ELISA analysis showed that activated Cry2Ab toxin could bind these two peptides with high affinity. The results indicate that employing this method can efficiently screen out target peptides with high affinity and specificity. The method also provides a valuable platform to discover the mode of other Bt toxins.

  6. Screening of p53 Promoter Binding Proteins by Phage Display in Liver Cells%筛选肝细胞p53启动子结合蛋白的噬菌体表面展示技术

    Institute of Scientific and Technical Information of China (English)

    吴顺华; 钟彦伟; 成军; 郑玉建

    2011-01-01

    目的 筛选p53启动子结合蛋白,探讨p53对肝细胞调节的分子生物学机制.方法 应用噬菌体表面展示技术,以p53作为固相筛选分子,对噬菌体人肝细胞cDNA文库进行5轮"吸附-洗脱-扩增"过程,经噬斑的PCR扩增后,构建克隆载体,最后对所筛选克隆进行DNA测序和生物信息学分析.结果 噬菌体经5轮富集后,成功构建了噬菌体p53展示文库,该文库包含与p53结合的肝细胞蛋白有17种.其中2个克隆编码未知功能蛋白;其余的克隆分别编码人α2HS糖蛋白(AHSG)、人D4、锌指蛋白家族2(DPF2)、凋亡反应锌指基因(REQ)、Ⅰ型纤溶酶原激活物抑制因子结合蛋白、氨基甲酰磷酸合成酶1、磷酸化酶激酶、酪氨酸氨基转移酶、酪蛋白激酶细胞周期素激酶、谷胱甘肽过氧化物酶、组蛋白结合蛋白3、跨膜蛋白2、血清黏蛋白2、有核红细胞白血病病毒癌基因2、毛细管扩张失调症突变基因,涉及到细胞发育和代谢、细胞外基质、细胞周期调控、信号转导、细胞凋亡以及原癌基因.结论 噬菌体表面展示技术成功筛选出了肝细胞p53启动子结合的多种类型和功能蛋白.%Objective To investigate the p53 regulation mechanisms in liver by screening binding proteins of p53 promoter using phage display. Methods Biotin p53 promoter DNA was incubated with a phage display cDNA library that expressed a library of human liver proteins on the surface of bacteriophage T7. Unbound phages were washed off and the phages bound to p53 were amplified. Positive plaqe were amplified by PCR and cloned into pGEM-Teasy vector in order to perform DNA sequence analysis and subsequently computer blasting analysis. Results Positive phage-displayed proteins binding with p53 promoter were enriched after five rounds of biopanning . Seventeen kinds of proteins might have relevance to p53,including Homo sapiens alpha-2-HS-glycoprotein (AHSG);Homo sapiens D4, zinc and double PHD

  7. Literature concerning control and display technology applicable to the Orbital Maneuvering Vehicle (OMV)

    Science.gov (United States)

    1990-01-01

    A review is presented of the literature concerning control and display technology that is applicable to the Orbital Maneuvering Vehicle (OMV), a system being developed by NASA that will enable the user to remotely pilot it during a mission in space. In addition to the general review, special consideration is given to virtual image displays and their potential for use in the system, and a preliminary partial task analysis of the user's functions is also presented.

  8. Double-Channel Stereoscopic Display Technology Based on OpenGL

    Directory of Open Access Journals (Sweden)

    Liu Shu

    2013-01-01

    Full Text Available The multi-channel stereoscopic display technology has become an attractive frontier research area in recent years, supporting users with correct spatial perception and large vision experience. This paper studies on the easiest case of double-channel. Focusing on the problem of complex network programming in traditional synchronous display method, a design plan of double-channel stereoscopic display system on one PC based on OpenGL is proposed, which reflects some innovation of the paper. A series of performance tests have been taken and the results agree well with the need for three-dimensional effect and synchronism.

  9. Engineered phages for electronics.

    Science.gov (United States)

    Cui, Yue

    2016-11-15

    Phages are traditionally widely studied in biology and chemistry. In recent years, engineered phages have attracted significant attentions for functionalization or construction of electronic devices, due to their specific binding, catalytic, nucleating or electronic properties. To apply the engineered phages in electronics, these are a number of interesting questions: how to engineer phages for electronics? How are the engineered phages characterized? How to assemble materials with engineered phages? How are the engineered phages micro or nanopatterned? What are the strategies to construct electronics devices with engineered phages? This review will highlight the early attempts to address these questions and explore the fundamental and practical aspects of engineered phages in electronics, including the approaches for selection or expression of specific peptides on phage coat proteins, characterization of engineered phages in electronics, assembly of electronic materials, patterning of engineered phages, and construction of electronic devices. It provides the methodologies and opens up ex-cit-ing op-por-tu-ni-ties for the development of a variety of new electronic materials and devices based on engineered phages for future applications.

  10. Exosome Display technology: applications to the development of new diagnostics and therapeutics.

    Science.gov (United States)

    Delcayre, Alain; Estelles, Angeles; Sperinde, Jeffrey; Roulon, Thibaut; Paz, Pedro; Aguilar, Barbara; Villanueva, Janeth; Khine, SuSu; Le Pecq, Jean-Bernard

    2005-01-01

    Exosome Display is a novel methodology enabling the manipulation of exosome protein content. This technology stems from the identification of addressing domains that mediate the specific distribution of proteins on exosomes. More particularly, Lactadherin expressed in non-mammary gland tissue has been found to localize to exosomes via binding of its C1C2 domain to exosome lipids. Exosome Display of soluble antigens and extracellular domains of membrane proteins that are not naturally found on exosomes occurs upon fusion of proteins with the Lactadherin C1C2 domain. Exosome Display of native full-length membrane proteins can also be achieved by non-restricted expression or sampling of membrane proteins on exosomes. These novel findings enable us to manipulate exosome composition and tailor exosomes with new desirable properties. The Exosome Display technology is very versatile since soluble, membrane-bound, trans-membrane or multimeric antigens that are not naturally found on exosomes can now be efficiently expressed at their surface in a native conformation. The technology was applied to the generation of antibodies against tumor biomarkers such as HLA/peptide complex. This antibody method called ExoMAb can be used to generate antibodies against any drug target candidates, notably including G-protein coupled receptors. The potential of Exosome Display technology for developing a broad range of novel diagnostics and therapeutics is discussed.

  11. Two novel neutralizing antigenic epitopes of the s1 subunit protein of a QX-like avian infectious bronchitis virus strain Sczy3 as revealed using a phage display peptide library.

    Science.gov (United States)

    Zou, Nianli; Xia, Jing; Wang, Fuyan; Duan, Zhenzhen; Miao, Dan; Yan, Qigui; Cao, Sanjie; Wen, Xintian; Liu, Ping; Huang, Yong

    2015-11-15

    The spike (S) protein of the infectious bronchitis virus (IBV) plays a central role in the pathogenicity, the immune antibody production, serotype and the tissue tropism. In this study, we generate 11 monoclonal antibodies (mAbs) against S1 subunit of IBV Sczy3 strain, and two mAbs 1D5 and 6A12 were positive in indirect ELISA against both His-S1 protein and the purified whole viral antigen. MAb 6A12 and 1D5 could recognized by other 10 IBV strains (IBVs) from five different genotypes, except that 1D5 had a relatively low reaction with two of the 10 tested IBVs. End-point neutralizing assay performed in chicken embro kidney (CEK) cells revealed that the neutralization titer of 6A12 and 1D5 against Sczy3 reached 1:44.7 and 1:40.6, respectively. After screening a phage display peptide library and peptide scanning, we identified two linear B-cell epitopes that were recognized by the mAbs 1D5 and 6A12, which corresponded to the amino acid sequences (87)PPQGMAW(93) and (412)IQTRTEP(418), respectively, in the IBV S1 subunit. Sequences comparison revealed that epitope (412)IQTRTEP(418) was conserved among IBVs, while the epitope (87)PPQGMAW(93) was relatively variable among IBVs. The novel mAbs and the epitopes identified will be useful for developing diagnostic assays for IBV infections.

  12. A strategy for the generation of specific human antibodies by directed evolution and phage display. An example of a single-chain antibody fragment that neutralizes a major component of scorpion venom.

    Science.gov (United States)

    Riaño-Umbarila, Lidia; Juárez-González, Victor Rivelino; Olamendi-Portugal, Timoteo; Ortíz-León, Mauricio; Possani, Lourival Domingos; Becerril, Baltazar

    2005-05-01

    This study describes the construction of a library of single-chain antibody fragments (scFvs) from a single human donor by individual amplification of all heavy and light variable domains (1.1 x 10(8) recombinants). The library was panned using the phage display technique, which allowed selection of specific scFvs (3F and C1) capable of recognizing Cn2, the major toxic component of Centruroides noxius scorpion venom. The scFv 3F was matured in vitro by three cycles of directed evolution. The use of stringent conditions in the third cycle allowed the selection of several improved clones. The best scFv obtained (6009F) was improved in terms of its affinity by 446-fold, from 183 nm (3F) to 410 pm. This scFv 6009F was able to neutralize 2 LD(50) of Cn2 toxin when a 1 : 10 molar ratio of toxin-to-antibody fragment was used. It was also able to neutralize 2 LD(50) of the whole venom. These results pave the way for the future generation of recombinant human antivenoms.

  13. Development of a workflow for screening and identification of α-amylase inhibitory peptides from food source using an integrated Bioinformatics-phage display approach: Case study - Cumin seed.

    Science.gov (United States)

    Siow, Hwee-Leng; Lim, Theam Soon; Gan, Chee-Yuen

    2017-01-01

    The main objective of this study was to develop an efficient workflow to discover α-amylase inhibitory peptides from cumin seed. A total of 56 unknown peptides was initially found in the cumin seed protein hydrolysate. They were subjected to 2 different in silico screenings and 6 peptides were shortlisted. The peptides were then subjected to in vitro selection using phage display technique and 3 clones (CSP3, CSP4 and CSP6) showed high affinity in binding α-amylase. These clones were subjected to the inhibitory test and only CSP4 and CSP6 exhibited high inhibitory activity. Therefore, these peptides were chemically synthesized for validation purposes. CSP4 exhibited inhibition of bacterial and human salivary α-amylases with IC50 values of 0.11 and 0.04μmol, respectively, whereas CSP6 was about 0.10 and 0.15μmol, respectively. Results showed that the strength of each protocol has been successfully combined as deemed fit to enhance the α-amylase inhibitor peptide discovery. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. 68Ga-labelled phage-display selected peptides as tracers for positron emission tomography imaging of Staphylococcus aureus biofilm-associated infections: Selection, radiolabelling and preliminary biological evaluation

    DEFF Research Database (Denmark)

    Nielsen, Karin M.; Kyneb, Majbritt H.; Alstrup, Aage Kristian Olsen

    2016-01-01

    stable in product formulation, whereas 68Ga-A8-K-DOTA was unstable. The S. aureus binding of 68Ga-A11-GSGK-DOTA and 68Ga-A9-K-DOTA was observed in vitro, though blocking of the binding was not possible by excess of cold peptide. The 68Ga-A9-K-DOTA was degraded slowly in vitro, while the combined in vivo......Introduction: In humans, Staphylococcus aureus is a major cause of skin and deep-sited infections, often associated with the formation of biofilms. The purpose of the study was to identify and 68Ga-label phage-display peptides selected against S. aureus biofilm and evaluate their potential......-N,N′,N″,N‴-tetraacetic acid (DOTA) conjugated via a lysine linker (K), and for A11 also a glycine-serine-glycine spacer (GSG). The 68Ga-labelling of A8-K-DOTA, A9-K-DOTA, and A11-GSGK-DOTA were optimized in regards to: pre-purification methods of generator eluates, labelling solution, time, and temperature, as well...

  15. Collection of phage-peptide probes for HIV-1 immunodominant loop-epitope.

    Science.gov (United States)

    Palacios-Rodríguez, Yadira; Gazarian, Tatiana; Rowley, Merrill; Majluf-Cruz, Abraham; Gazarian, Karlen

    2007-02-01

    Early diagnosis and prevention of human immunodeficiency virus type-1 (HIV-1) infection, which remains a serious public health threat, is inhibited by the lack of reagents that elicit antiviral responses in the immune system. To create mimotopes (peptide models of epitopes) of the most immunodominant epitope, CSGKLIC, that occurs as a loop on the envelope gp41 glycoprotein and is a key participant in infection, we used phage-display technology involving biopanning of large random libraries with IgG of HIV-1-infected patients. Under the conditions used, library screening with IgG from patient serum was directed to the CSGKLIC epitope. Three rounds of selection converted a 12 mer library of 10(9) sequences into a population in which up to 79% of phage bore a family of CxxKxxC sequences ("x" designates a non-epitope amino acid). Twenty-one phage clones displaying the most frequently selected peptides were obtained and were shown to display the principal structural (sequence and conformational), antigenic and immunogenic features of the HIV-1 immunodominant loop-epitope. Notably, when the mixture of the phage mimotopes was injected into mice, it induced 2- to 3-fold higher titers of antibody to the HIV-1 epitope than could be induced from individual mimotopes. The described approach could be applicable for accurately reproducing HIV-1 epitope structural and immunological patterns by generation of specialized viral epitope libraries for use in diagnosis and therapy.

  16. Touch displays: the effects of palm rejection technology on productivity, comfort, biomechanics and positioning.

    Science.gov (United States)

    Camilleri, Matt J; Malige, Ajith; Fujimoto, Jeffrey; Rempel, David M

    2013-01-01

    Direct touch displays can improve the human-computer experience and productivity; however, the higher hand locations may increase shoulder fatigue. Palm rejection (PR) technology may reduce shoulder loads by allowing the palms to rest on the display and increase productivity by registering the touched content and fingertips through the palms rather than shoulders. The effects of PR were evaluated by having participants perform touch tasks while posture and reaction force on the display were measured. Enabling PR, during which the subjects could place the palms on the display (but were not required to), resulted in increased wrist extension, force applied to the display and productivity, and less discomfort, but had no effect on the self-selected positioning of the display. Participants did not deliberately place their palms on the display; therefore, there was no reduction in shoulder load and the increased productivity was not due to improved hand registration. The increased productivity may have been due to reduced interruptions from palm contacts or reduced motor control demands.

  17. Gaze directed displays as an enabling technology for attention aware systems

    NARCIS (Netherlands)

    Toet, A.

    2006-01-01

    Visual information can in principle be dynamically optimised by monitoring the user’s state of attention, e.g. by tracking eye movements. Gaze directed displays are therefore an important enabling technology for attention aware systems. We present a state-of-the-art review of both (1) techniques to

  18. 77 FR 5865 - American Unity Investments, Inc., China Display Technologies, Inc., China Wind Energy, Inc., Fuda...

    Science.gov (United States)

    2012-02-06

    ... From the Federal Register Online via the Government Publishing Office ] SECURITIES AND EXCHANGE COMMISSION American Unity Investments, Inc., China Display Technologies, Inc., China Wind Energy, Inc., Fuda... current and accurate information concerning the securities of China Wind Energy, Inc. because it has...

  19. Construction and characterization of a non-immune Llama variable heavy chain phage display antibody library%羊驼非免疫重链单域抗体库的构建和鉴定

    Institute of Scientific and Technical Information of China (English)

    吴标; 王树军; 夏立亮; 季萍; 葛海良; 赵国屏; 王颖

    2011-01-01

    本研究旨在通过构建羊驼非免疫重链单域抗体库,完成抗体库多样性的鉴定,为进一步筛选抗原特异性重链抗体奠定基础.我们从未经免疫的羊驼外周血中分离外周血单个核细胞(PBMC),抽提RNA后,用RT-PCR方法特异性扩增羊驼重链抗体可变区(VHH)片段;并采用两步连接方法将重链抗体可变区片段与噬菌粒载体pCANTAB5E连接获得重组子,多次电转感受态大肠杆菌TG1后获得VHH抗体基因库;并采用稀释计数法测定抗体库库容量,随机挑取克隆测序验证抗体库多样性.结果显示,我们所构建的羊驼非免疫重链单域抗体库的库容量为1.5×109,随机克隆测序验证多样性良好,独立克隆所占比例为80%,并显示出和人源抗体较高的同源性.上述结果表明,我们已经成功构建获得大容量的羊驼非免疫重链单域抗体库,为进一步筛选抗原特异性重链抗体奠定基础.%To construct a non-immune Llama variable domain of heavy chain antibody(VHH) phage display antibody library (VHH antibody library). Llama peripheral blood mononuclear lymphocytes were isolated from whole blood by Ficoll-hypaque density gradient centrifugation. Total RNA was extracted from PBMCs and reverse-transcripted into cDNA by using specific primers. VHH were amplified by nested PCR. PCR products of VHH fragments were then purified and ligated with phagemid vector pCANTAB5E by a modified two-step ligation method. Recombinant pCANTAB5E-VHH vectors were electroporated into competent TGI E.coli cells to obtain the primary VHH antibody library. The library capacity was titrated through limited dilution. Recombination efficacy and diversity of VHH antibody library was determined by sequencing analysis. Alignment a-nalysis was performed to compare the homology between Llama VHH domain and human/mouse variable regions of heavy chains. By using a modified strategy, we have constructed a non-immune Llama VHH antibody library with 1. 5

  20. Progress of phage antibody library technique and its application prospect in aquaculture%噬菌体抗体库技术及其在水产养殖的应用前景

    Institute of Scientific and Technical Information of China (English)

    章晋勇; 吴英松; 汪建国

    2004-01-01

    Phage display antibody library has been proven to be a powerful technique used in development of antibodies. Since it was established in 1990, the technology has made enormous improvement and played more and more important role in basic research of biology, immunology, oncology, protein engineering, ligand-receptor studies and proteomics among others in last two decades while there is no report about the application of it in aquaculture so far. It is a success implication of phage display technique in antibody engineering in which antibodies or antibody fragments are displayed on the surface of filamentous bacteriophage by genetic fusion to a coat protein of phage. Cooperating with the effective screening technique, affinity panning, these form the principle of phage display antibody library. The most characteristic of it is a direct physical link between phenotype and genotype. So,the technology makes it practicable to improve characteristic of selected antibodies by genetic manipulation. In the present work, the background, principle and advantages of the powerful tool over traditional hybridroma technolgy are summarized. In addition, several key problems possible to face in the course of application of the technology,including improving the diversity of library, augmentation of library size, generation of high affinity antibody and effective screening of specific antibody were dissertated. At last the possible implication prospect of phage display antibody technique in aquaculture was discussed, especially in elucidating the immune system of fish and producing large amount antibodies with important diagnostic and therapeutic value in fish diseases.

  1. Bacteria, phages and septicemia.

    Directory of Open Access Journals (Sweden)

    Ausra Gaidelyte

    Full Text Available The use of phages is an attractive option to battle antibiotic resistant bacteria in certain bacterial infections, but the role of phage ecology in bacterial infections is obscure. Here we surveyed the phage ecology in septicemia, the most severe type of bacterial infection. We observed that the majority of the bacterial isolates from septicemia patients spontaneously secreted phages active against other isolates of the same bacterial strain, but not to the strain causing the disease. Such phages were also detected in the initial blood cultures, indicating that phages are circulating in the blood at the onset of sepsis. The fact that most of the septicemic bacterial isolates carry functional prophages suggests an active role of phages in bacterial infections. Apparently, prophages present in sepsis-causing bacterial clones play a role in clonal selection during bacterial invasion.

  2. 三维立体显示技 术%Three Dimensional Display Technology

    Institute of Scientific and Technical Information of China (English)

    王爱红; 王琼华; 李大海; 陶宇虹; 赵仁亮; 赵悟翔

    2008-01-01

    实现三维立体显示是人类长期的梦想.三维立体显示技术已成为当今一个引人注目的前沿科技领域.文中介绍了三维立体显示的概念和一些技术方法.自由立体显示技术是当今立体显示的主流技术,文中阐述了该技术的研发动态.最后概述了三维立体显示的重要应用及意义.%The realization of three dimensional (3D) display is a long fostered dream. 3D display becomes an attractive frontier research area. The concepts, some technologies about 3D display are expounded. The states of the art for two mainstream technologies of auto-stereoscopic 3D display are presented. The impor-tant applications of 3D display are briefly given.

  3. Heat tolerance of dairy lactococcal c2 phages

    DEFF Research Database (Denmark)

    Nielsen, Cecilie Lykke Marvig; Basheer, Aideh; Neve, H.

    2011-01-01

    Nine Lactococcus lactis c2 phages propagated on different hosts were screened for thermal resistance in skimmed milk. Pronounced variations in thermal resistance were found. Three phages displayed high sensitivity towards heat resulting in >8 log reductions after 70 °C for 5 min, whereas the most...

  4. Screening, identification and significance of the phage-display random 7 amino acid peptide specific to the sera of patients with systemic lupus erythematosus%系统性红斑狼疮患者血清特异性的噬菌体7肽的筛选、鉴定及其意义

    Institute of Scientific and Technical Information of China (English)

    王垚; 钟照华; 张凤民; 苏丽菊; 李辉; 刘彦虹; 翟爱霞; 考文萍; 吴静; 李文辉; 胡云龙

    2010-01-01

    Objective To screen and identify the phage-display random 7 amino acid peptide specific to the systemic lupus erythematosus(SLE) and analyze its practical significance. Methods Using the phage random 7 peptide library screening, the SLE specific phage clones are obtained after binding with the mixture of sera from 30 SLE patients and 30 normal controls as ligand respectively. Then the Dot-ELISA is used to identify the SLE specific phage clones reactive to sera of the SLE patients and normal controls individually. Finally the identified phage-display random 7 amino acid peptides are sequenced and it's homology with the antigenic epitope of human being and other are also analyzed. Results Total 12 of the phage-display random 7 amino acid peptide are obtained by phage peptide library screening and the Dot-ELISA identification. Sequence analysis shows that the identified phage-display random 7 amino acid peptide epitope have homology with E. coli, Salmonella and human immunodeficiency virus, but not with that of human being. Conclusion SLE-specific peptides screened by phage random peptide library maybe used to diagnosis the SLE. Meanwhile, the antibodies in SLE patients which are combined with the Pathogen epitope, suggest that SLE maybe relate to pathogen infection.%目的 筛选和鉴定与系统性红斑狼疮(systemic lupus erythematosus,SLE)患者血清特异性结合的噬菌体7肽,并分析其实际意义.方法 分别选取正常人及SLE患者血清各30例,先后用正常人混合血清及SLE患者混合血清作为筛选配基,对噬菌体随机7肽库进行亲和筛选、扩增,获得SLE血清特异性结合的噬菌体克隆,并用患者混合血清进行Dot-ELISA实验鉴定获得的噬菌体克隆,进而分别用SLE患者及正常人血清各12例进一步鉴定阳性噬菌体的混合克隆,确定阳性噬菌体克隆与个体血清之间的结合情况;并对最终鉴定的噬菌体克隆进行测序与比对分析.结果 筛选到与SLE患者混合

  5. ProtEx: a novel technology to display exogenous proteins on the cell surface for immunomodulation.

    Science.gov (United States)

    Singh, Narendra P; Yolcu, Esma S; Askenasy, Nadir; Shirwan, Haval

    2005-11-01

    Gene therapy as an immunomodulatory approach has the potential to treat various inherited and acquired immune-based human diseases. However, its clinical application has several challenges, varying from the efficiency of gene transfer, control of gene expression, cell and tissue targeting, and safety concerns associated with the introduction of exogenous DNA into cells/tissues. Gene therapy is also a time- and labor-intensive procedure. As an alternative, we recently developed a novel technology, ProtEx, that allows for rapid, efficient, and durable display of exogenous proteins on the surface of cells, tissues, and organs without detectable toxicity. This technology exploits the strong binding affinity (Kd = 10(-15) M) of streptavidin with biotin and involves generation of chimeric molecules composed of the extracellular portions of immunological proteins of interest and a modified form of streptavidin, biotinylation of biological surfaces, and decoration of the modified surface with chimeric proteins. Biotin persists on the cell surface for weeks both in vitro and in vivo, thereby providing a platform to display exogenous proteins with extended cell surface kinetics. Two chimeric proteins, rat FasL (SA-FasL) and human CD80 (CD80-SA), were generated and tested for cell surface display and immunomodulatory functions. SA-FasL and CD80-SA molecules persisted on the surface of various cell types for extended periods, varying from days to weeks in vitro and in vivo. The cell surface kinetics, however, were protein and cell type dependent. SA-FasL showed potent apoptotic activity against Fas+ cells as a soluble protein or displayed on the cell surface and effectively blocked alloreactive responses. The display of CD80-SA on the surface of tumor cells, however, converted them into antigen-presenting cells for effective stimulation of autologous and allogeneic T-cell responses. ProtEx technology, therefore, represents a practical and effective alternative to DNA

  6. Ajax, XSLT and SVG: Displaying ATLAS conditions data with new web technologies

    CERN Document Server

    Roe, S A

    2010-01-01

    The combination of three relatively recent technologies is described which allows an easy path from database retrieval to interactive web display. SQL queries on an Oracle database can be performed in a manner which directly return an XML description of the result, and Ajax techniques (Asynchronous JavaScript And XML) are used to dynamically inject the data into a web display accompanied by an XSLT transform template which determines how the data will be formatted. By tuning the transform to generate SVG (Scalable Vector Graphics) a direct graphical representation can be produced in the web page while retaining the database data as the XML source, allowing dynamic links to be generated in the web representation, but programmatic use of the data when used from a user application. With the release of the SVG 1.2 Tiny draft specification, the display can also be tailored for display on mobile devices. The technologies are described and a sample application demonstrated, showing conditions data from the ATLAS Sem...

  7. Rapid enumeration of phage in monodisperse emulsions.

    Science.gov (United States)

    Tjhung, Katrina F; Burnham, Sean; Anany, Hany; Griffiths, Mansel W; Derda, Ratmir

    2014-06-17

    Phage-based detection assays have been developed for the detection of viable bacteria for applications in clinical diagnosis, monitoring of water quality, and food safety. The majority of these assays deliver a positive readout in the form of newly generated progeny phages by the bacterial host of interest. Progeny phages are often visualized as plaques, or holes, in a lawn of bacteria on an agar-filled Petri dish; however, this rate-limiting step requires up to 12 h of incubation time. We have previously described an amplification of bacteriophages M13 inside droplets of media suspended in perfluorinated oil; a single phage M13 in a droplet yields 10(7) copies in 3-4 h. Here, we describe that encapsulation of reporter phages, both lytic T4-LacZ and nonlytic M13, in monodisperse droplets can also be used for rapid enumeration of phage. Compartmentalization in droplets accelerated the development of the signal from the reporter enzyme; counting of "positive" droplets yields accurate enumeration of phage particles ranging from 10(2) to 10(6) pfu/mL. For enumeration of T4-LacZ phage, the fluorescent signal appeared in as little as 90 min. Unlike bulk assays, quantification in emulsion is robust and insensitive to fluctuations in environmental conditions (e.g., temperature). Power-free emulsification using gravity-driven flow in the absence of syringe pumps and portable fluorescence imaging solutions makes this technology promising for use at the point of care in low-resource environments. This droplet-based phage enumeration method could accelerate and simplify point-of-care detection of the pathogens for which reporter bacteriophages have been developed.

  8. Use of micro-emulsion technology for the directed evolution of antibodies.

    Science.gov (United States)

    Buhr, Diane L; Acca, Felicity E; Holland, Erika G; Johnson, Katie; Maksymiuk, Gail M; Vaill, Ada; Kay, Brian K; Weitz, David A; Weiner, Michael P; Kiss, Margaret M

    2012-09-01

    Affinity reagents, such as antibodies, are needed to study protein expression patterns, sub-cellular localization, and post-translational modifications in complex mixtures and tissues. Phage Emulsion, Secretion, and Capture (ESCape) is a novel micro-emulsion technology that utilizes water-in-oil (W/O) emulsions for the identification and isolation of cells secreting phage particles that display desirable antibodies. Using this method, a large library of antibody-displaying phage will bind to beads in individual compartments. Rather than using biopanning on a large mixed population, phage micro-emulsion technology allows us to individually query clonal populations of amplified phage against the antigen. The use of emulsions to generate microdroplets has the promise of accelerating phage selection experiments by permitting fine discrimination of kinetic parameters for binding to targets. In this study, we demonstrate the ability of phage micro-emulsion technology to distinguish two scFvs with a 300-fold difference in binding affinities (100nM and 300pM, respectively). In addition, we describe the application of phage micro-emulsion technology for the selection of scFvs that are resistant to elevated temperatures. Copyright © 2012. Published by Elsevier Inc.

  9. Application of high-definition display technology to the design of mobile client/server systems

    Science.gov (United States)

    Johnson, Roger L.; Williams, D. E.

    1994-04-01

    Although the major market force behind the development of high definition display systems is consumer television, there are also a number of important applications for this technology in computer systems which are being designed for the transportation, manufacturing, field engineering, maintenance, and scientific research industries. In many of these applications the computer workstations will need to be highly mobile (i.e. easily transported and often hand carried) and will require flat panel, projection, and virtual image high definition displays to provide graphics and imagery to the user at the point of action. One such important area of endeavor which includes all of the above applications is the international manned space exploration and science program. Current research work underway at SAIC is focused on the development of advanced mobile computing systems which utilize high definition displays. These systems are being specifically designed to support the user in the remote field environments anticipated by the space exploration program. This paper provides a view of future utilization of high definition displays and mobile computing systems in the remote field environments associated with the manned space program. The presentation will illustrate how the development of these systems can be used to greatly improve worker efficiency through the concept of telepresence.

  10. Preferential germline usage and VH/VL pairing observed in human antibodies selected by mRNA display.

    Science.gov (United States)

    Chen, Lei; Kutskova, Yuliya A; Hong, Feng; Memmott, John E; Zhong, Suju; Jenkinson, Megan D; Hsieh, Chung-Ming

    2015-10-01

    Since the invention of phage display, in vitro antibody display technologies have revolutionized the field of antibody discovery. In combination with antibody libraries constructed with sequences of human origin, such technologies enable accelerated therapeutic antibody discovery while bypassing the laborious animal immunization and hybridoma generation processes. Many in vitro display technologies developed since aim to differentiate from phage display by displaying full-length IgG proteins, utilizing eukaryotic translation system and codons, increasing library size or real-time kinetic selection by fluorescent activated cell sorting. We report here the development of an mRNA display technology and an accompanying HCDR3 size spectratyping monitor for human antibody discovery. Importantly, the mRNA display technology maintains a monovalent linkage between the mRNA (genotype) and display binding protein (phenotype), which minimizes avidity effect common in other display systems and allows for a stringent affinity and off-rate selection. The mRNA display technology successfully identified 100 human antibodies in 15 different selections against various targets from naïve human antibody libraries. These antibodies in general have high affinity and diversity. By analyzing the germline usage and combination of antibodies selected by the mRNA display technology, we identified trends and determined the productivity of each germline subgroup in the libraries that could serve as the knowledge base for constructing fully synthetic, next generation antibody libraries.

  11. Synthetic Vision System Commercial Aircraft Flight Deck Display Technologies for Unusual Attitude Recovery

    Science.gov (United States)

    Prinzel, Lawrence J., III; Ellis, Kyle E.; Arthur, Jarvis J.; Nicholas, Stephanie N.; Kiggins, Daniel

    2017-01-01

    A Commercial Aviation Safety Team (CAST) study of 18 worldwide loss-of-control accidents and incidents determined that the lack of external visual references was associated with a flight crew's loss of attitude awareness or energy state awareness in 17 of these events. Therefore, CAST recommended development and implementation of virtual day-Visual Meteorological Condition (VMC) display systems, such as synthetic vision systems, which can promote flight crew attitude awareness similar to a day-VMC environment. This paper describes the results of a high-fidelity, large transport aircraft simulation experiment that evaluated virtual day-VMC displays and a "background attitude indicator" concept as an aid to pilots in recovery from unusual attitudes. Twelve commercial airline pilots performed multiple unusual attitude recoveries and both quantitative and qualitative dependent measures were collected. Experimental results and future research directions under this CAST initiative and the NASA "Technologies for Airplane State Awareness" research project are described.

  12. Wireless communication technology as applied to head mounted display for a tactical fighter pilot

    Science.gov (United States)

    Saini, Gurdial S.

    2007-04-01

    The use of Helmet-Mounted Display/Tracker (HMD/Ts) is becoming widespread for air-to-air, within visual range target acquisition for a tactical fighter pilot. HMD/Ts provide the aircrew with a significant amount of information on the helmet, which reduces the burden of the aircrew from having to continually look down in the cockpit to receive information. HMD/Ts allow the aircrew to receive flight and targeting information regardless of line-of-sight, which should increase the aircrew's situation awareness and mission effectiveness. Current technology requires that a pilot wearing a Helmet Mounted Display/Tracker be connected to the aircraft with a cable. The design of this cable is complex, costly, and its use can decrease system reliability. Most of the problems associated with the use of cable can be alleviated by using wireless transmission for all signals. This will significantly reduce or eliminate the requirements of the interconnect cable/connector reducing system complexity, and cost, and enhancing system safety. A number of wireless communication technologies have been discussed in this paper and the rationale for selecting one particular technology for this application has been shown. The problems with this implementation and the direction of the future effort are outlined.

  13. 利用噬菌体随机9肽库筛选寻常型天疱疮抗原模拟表位%Selection of mimotopes of pemphigus vulgaris antigen from a phage-displayed random nonapeptide library

    Institute of Scientific and Technical Information of China (English)

    黄丽群; 姚刚; 薛峰; 潘萌; 孙兵; 郑捷

    2008-01-01

    目的 利用噬菌体随机9肽库筛选寻常型天疱疮抗原桥粒芯糖蛋白3(desmoglein,Dsg3)模拟表位,加深对寻常型天疱疮发病机制的认识. 方法 通过大肠杆菌表达Dsg3的胞外结构域1和2(extracellular domain,EC1-2)和谷胱甘肽转移酶(GST)的融合蛋白,从寻常型天疱疮患者血清中纯化与EC1-2特异结合的多克隆抗体,对噬菌体随机线九肽库和环九肽库进行亲和筛选,阳件噬菌体展示肽经ELISA和竞争性ELISA验证. 结果 经过两轮亲和筛选,与自身抗体结合的噬菌体明显富集,免疫筛查、验证后得到3个阳性噬菌体展示肽,ELISA检测显示它们与患者血清反应,而不与正常人血清反应,竞争性ELISA检测显示它们可以抑制寻常型天疱疮患者血清与重组蛋白EC1-2的结合. 结论 利用噬菌体随机9肽库筛选到3个与寻常性大疱疮密切相关的模拟表位.%Objective To screen the mimotopes ofpemphigus vulgaris (PV) antigen, desmoglein3 (Dsg3) with a phage-displayed random nonapeptide library, so as to update the knowledge on the patho-genesis of PV. Methods Recombinant fusion protein of extracellular domain 1-2 (EC1-2) of Dsg3 and glutathione transferase was expressed by E.coli BL21, and used to purify polyclonal autoantibody binding to recombinant EC 1-2 from the sera of patients with PV. Then, selected autoantibody was applied as a ligand for biopanning of a phage-displayed linear random nonapeptide library and circular random nonapeptide library. Monoclonal phages were selected by immunoscreening and tested with ELISA and competitive ELISA. Results After two rounds ofbiopanning, a population ofpeptide-displaying phages binding to autoan- tidody were highly enriched. Sixty individual phage clones selected by immunosereening were further sub-jected to screening with ELISA and competitive ELISA. Finally, three positive phage clones were obtained. As shown by ELISA and competitive ELISA, they reacted with serum from

  14. High-throughput Identification of Phage-derived Imaging Agents

    Directory of Open Access Journals (Sweden)

    Kimberly A. Kelly

    2006-01-01

    Full Text Available The use of phage-displayed peptide libraries is a powerful method for selecting peptides with desired binding properties. However, the validation and prioritization of “hits” obtained from this screening approach remains challenging. Here, we describe the development and testing of a new analysis method to identify and display hits from phage-display experiments and high-throughput enzyme-linked immunosorbent assay screens. We test the method using a phage screen against activated macrophages to develop imaging agents with higher specificity for active disease processes. The new methodology should be useful in identifying phage hits and is extendable to other library screening methods such as small-molecule and nanoparticle libraries.

  15. Comparative genomic analysis of Pseudomonas aeruginosa phage PaMx25 reveals a novel siphovirus group related to phages infecting hosts of different taxonomic classes.

    Science.gov (United States)

    Flores, Víctor; Sepúlveda-Robles, Omar; Cazares, Adrián; Kameyama, Luis; Guarneros, Gabriel

    2017-08-01

    Bacteriophages (phages) are estimated to be the most abundant and diverse entities in the biosphere harboring vast amounts of novel genetic information. Despite the genetic diversity observed, many phages share common features, such as virion morphology, genome size and organization, and can readily be associated with clearly defined phage groups. However, other phages display unique genomes or, alternatively, mosaic genomes composed of regions that share homology with those of phages of diverse origins; thus, their relationships cannot be easily assessed. In this work, we present a functional and comparative genomic analysis of Pseudomonas aeruginosa phage PaMx25, a virulent member of the Siphoviridae family. The genomes of PaMx25 and a highly homologous phage NP1, bore sequence homology and synteny with the genomes of phages that infect hosts different than Pseudomonas. In order to understand the relationship of the PaMx25 genome with that of other phages, we employed several computational approaches. We found that PaMx25 and NP1 effectively bridged several phage groups. It is expected that as more phage genomes become available, more gaps will be filled, blurring the boundaries that currently separate phage groups.

  16. Optical gesture sensing and depth mapping technologies for head-mounted displays: an overview

    Science.gov (United States)

    Kress, Bernard; Lee, Johnny

    2013-05-01

    Head Mounted Displays (HMDs), and especially see-through HMDs have gained renewed interest in recent time, and for the first time outside the traditional military and defense realm, due to several high profile consumer electronics companies presenting their products to hit market. Consumer electronics HMDs have quite different requirements and constrains as their military counterparts. Voice comments are the de-facto interface for such devices, but when the voice recognition does not work (not connection to the cloud for example), trackpad and gesture sensing technologies have to be used to communicate information to the device. We review in this paper the various technologies developed today integrating optical gesture sensing in a small footprint, as well as the various related 3d depth mapping sensors.

  17. Bridging the gap: adapting advanced display technologies for use in hybrid control rooms

    Energy Technology Data Exchange (ETDEWEB)

    Jokstad, Håkon [Inst. for Energy Technology, Halden (Norway); Boring, Ronald [Idaho National Lab. (INL), Idaho Falls, ID (United States)

    2015-02-01

    recently assisted INL in establishing the technical infrastructure for implementation of HSI prototypes from HAMMLAB into the HSSL to demonstrate relevant control room replacement systems in support of the LWRS program. In March, 2014, IFE delivered the first HSI prototype utilizing this infrastructure — a large screen overview display for INL's simulator. The co-operation now continues by developing Procedure Support Displays targeted for operators in hybrid control room settings. These prototypes are being validated with U.S. reactor operators in the HSSL and optimized to enhance their performance. This research serves as a crucial stepping stone toward incorporation of advanced display technologies into conventional main control rooms.

  18. 应用噬菌体肽库技术获得与猪鼻支原体蛋白P37结合的多肽序列%Identification of Oligopeptides Binding to Mycoplasma Hyorhinis P37 Using a Phage Display Library

    Institute of Scientific and Technical Information of China (English)

    杨华; 冯勤; 徐慧玉; 寿成超

    2011-01-01

    Phage display random heptapeptide library was screened with recombinant P37 in this study. The positive phage clones were identified by ELISA and were sequenced, and the amino acid sequences of the polypeptides displayed on phage were deduced. After GST-polypeptides fusion protein was constructed and expressed, its binding to P37 was determined by GST-pull down and Western blot. After 4 rounds of bio-panning, the enriched positive phage clones were identified by ELISA. Eighteen positive phage clones were sequenced and the peptide sequences were as follows, ACAPKPPWLC (12/18), RPLSIDPWSPHL (3/18), RPLSNDPWSPHL (1/18), QNMMSPIEGVRI (1/ 18) and WAPEKDYMQLMK (1/18). The results from GST-pull down and Western blot showed that peptide RPLSIDPWSPHL could interact with P37. The study will be helpful for identifying the protein reacting with P37.%本研究以猪鼻支原体P37蛋白作为筛选分子,对噬菌体展示随机肽库进行亲和淘选.利用ELISA法鉴定亲和力高的阳性噬菌体克隆,对其进行DNA测序和分析,据此推导随机多肽的氨基酸序列,然后构建、表达GST-多肽重组蛋白,经GST-pull down进一步鉴定该多肽与P37的结合力.经过4轮筛选和单克隆检测,获得18个有较强特异反应的阳性克隆,18个克隆包括了5种不同的小肽序列,它们分别为ACAPKPPWLC(12/18)、RPLSIDP-WSPHL(3/18)、RPLSNDPWSPHL(1/18)、QNMMSPIEGVRI (1/18)和WAPEKDYMQLMK(1/18).用ACAPKPPWLC、RPLSIDPWSPHL与GST重组的融合蛋白进行GST-pull down实验表明,RPLSIDPWSPHL多肽能与P37相互作用.本研究为进一步筛选与P37相互作用的蛋白提供了实验依据和结构基础.

  19. Phage therapy: present and future

    Science.gov (United States)

    Kolesnikova, S. G.; Tulyakova, E. N.; Moiseeva, I. Y.

    2017-01-01

    In recent years, bacteriophages are known to have become an effective alternative to antibiotic drugs. The article describes the current and potential applications of bacteriophages and phage endolysins. Also of interest is the devastating effect of phages on biofilms. The development of phage resistance is touched upon as well. Furthermore, the authors discuss the issue of laying down the rules of rational phage therapy.

  20. Estimating richness from phage metagenomes

    Science.gov (United States)

    Bacteriophages are important drivers of ecosystem functions, yet little is known about the vast majority of phages. Phage metagenomics, or the study of the collective genome of an assemblage of phages, enables the investigation of broad ecological questions in phage communities. One ecological cha...

  1. 一种特异性识别斑马鱼卵黄蛋白原的噬菌体展示单链抗体的可溶性表达方案%Strategy for soluble expression of phage-displayed scFv antibody specific for zebrafish vitellogenin

    Institute of Scientific and Technical Information of China (English)

    饶瑜; 钟利桥; 张凤; 张晓华; 戴和平

    2011-01-01

    We developed a new method for soluble expression of phage-displayed scFv antibody specific for zebrafish vitellogenin. The scFv antibody F5 could bind zebrafish vitellogenin specifically in phage-displayed form but not soluble form. The gene of scFv antibody F5 was cloned into vector pET 32a and transferred into Escherichia coli ori DE3. With inducible expression, soluble scFv antibody 32a-F5 was obtained successfully and could also specifically bind to zebrafish vitellogenin. The insoluble expression of phage-displayed scFv antibody was a common problem in the practical use of phage display. This study offered a feasible way to express soluble scFv antibodies with biological activity.%为了实现特异性识别斑马鱼卵黄蛋白原的噬菌体展示单链抗体的可溶性表达,将不能以可溶性蛋白形式表达的、只能以噬菌体展示形式特异性识别斑马鱼卵黄蛋白原的单链抗体F5的基因,克隆到pET 32a载体并转化入大肠杆菌ori DE3中.结果表明,通过诱导表达,可获得可溶性的并且仍特异性识别斑马鱼卵黄蛋白原的单链抗体32a-F5.噬菌体展示单链抗体不能可溶性表达是噬菌体展示技术应用中常见的问题,该方法提供了一种表达可溶性单链抗体的可行性方案.

  2. Isolation and characterization of numerous novel phages targeting diverse strains of the ubiquitous and opportunistic pathogen Achromobacter xylosoxidans.

    Science.gov (United States)

    Wittmann, Johannes; Dreiseikelmann, Brigitte; Rohde, Christine; Rohde, Manfred; Sikorski, Johannes

    2014-01-01

    The clinical relevance of nosocomially acquired infections caused by multi-resistant Achromobacter strains is rapidly increasing. Here, a diverse set of 61 Achromobacter xylosoxidans strains was characterized by MultiLocus Sequence Typing and Phenotype MicroArray technology. The strains were further analyzed in regard to their susceptibility to 35 antibiotics and to 34 different and newly isolated bacteriophages from the environment. A large proportion of strains were resistant against numerous antibiotics such as cephalosporines, aminoglycosides and quinolones, whereas piperacillin-tazobactam, ticarcillin, mezlocillin and imipenem were still inhibitory. We also present the first expanded study on bacteriophages of the genus Achromobacter that has been so far a blank slate with respect to phage research. The phages were isolated mainly from several waste water treatment plants in Germany. Morphological analysis of all of these phages by electron microscopy revealed a broad diversity with different members of the order Caudovirales, including the families Siphoviridae, Myoviridae, and Podoviridae. A broad spectrum of different host ranges could be determined for several phages that lysed up to 24 different and in part highly antibiotic resistant strains. Molecular characterisation by DNA restriction analysis revealed that all phages contain linear double-stranded DNA. Their restriction patterns display distinct differences underlining their broad diversity.

  3. Isolation and characterization of numerous novel phages targeting diverse strains of the ubiquitous and opportunistic pathogen Achromobacter xylosoxidans.

    Directory of Open Access Journals (Sweden)

    Johannes Wittmann

    Full Text Available The clinical relevance of nosocomially acquired infections caused by multi-resistant Achromobacter strains is rapidly increasing. Here, a diverse set of 61 Achromobacter xylosoxidans strains was characterized by MultiLocus Sequence Typing and Phenotype MicroArray technology. The strains were further analyzed in regard to their susceptibility to 35 antibiotics and to 34 different and newly isolated bacteriophages from the environment. A large proportion of strains were resistant against numerous antibiotics such as cephalosporines, aminoglycosides and quinolones, whereas piperacillin-tazobactam, ticarcillin, mezlocillin and imipenem were still inhibitory. We also present the first expanded study on bacteriophages of the genus Achromobacter that has been so far a blank slate with respect to phage research. The phages were isolated mainly from several waste water treatment plants in Germany. Morphological analysis of all of these phages by electron microscopy revealed a broad diversity with different members of the order Caudovirales, including the families Siphoviridae, Myoviridae, and Podoviridae. A broad spectrum of different host ranges could be determined for several phages that lysed up to 24 different and in part highly antibiotic resistant strains. Molecular characterisation by DNA restriction analysis revealed that all phages contain linear double-stranded DNA. Their restriction patterns display distinct differences underlining their broad diversity.

  4. Phage choice, isolation, and preparation for phage therapy.

    Science.gov (United States)

    Gill, Jason J; Hyman, Paul

    2010-01-01

    Phage therapy is the use of bacteriophages--viruses that use bacteria as their host cells--as biocontrol agents of bacteria. Currently, phage therapy is garnering renewed interest as bacterial resistance to antibiotics becomes widespread. Historically, phage therapy was largely abandoned in the West in the 1940s due to the advent of chemical antibiotics, and the unreliability of phage-based treatments when compared to antibiotics. The choice of phage strain and the methods of phage preparation are now thought to have been critical to the success or failure of phage therapy trials. Insufficiently virulent phages, especially against actual target bacteria, allow bacteria to survive treatment while poorly prepared phage stocks, even if of sufficiently virulent phages, lack the numbers of viable phages required for adequate treatment. In this review we discuss the factors that determine the methods of isolation, analysis, and identification of phage species for phage therapy. We go on to discuss the various methods available for purifying phages as well as considerations of the degree of purification which is sufficient for various applications. Lastly, we review the current practices used to prepare commercial phage therapy products.

  5. Detection of sulfur mustard adducts in human callus by phage antibodies

    NARCIS (Netherlands)

    Bikker, F.J.; Mars-Groenendijk, R.H.; Noort, D.; Fidder, A.; Schans, G.P. van der

    2007-01-01

    As part of a research program to develop novel methods for diagnosis of sulfur mustard exposure in the human skin the suitability of phage display was explored. Phage display is a relative new method that enables researchers to quickly evaluate a huge range of potentially useful antibodies, thereby

  6. Selection of Triptolide Ligands from a Random Phage Display Library and Primary Verification of Their Combination%雷公藤内酯醇靶蛋白的筛选及其结合的初步验证

    Institute of Scientific and Technical Information of China (English)

    杨旭光; 徐晓煜; 李家璜; 姚其正; 朱彤阳; 华子春; 郑伟娟

    2012-01-01

    Triptolide is an extract from the Chinese herb Tripterygium wilfordii Hook f. We screened out a peptide from a C7 phage display library and presumed it was a potential peptide ligand for triptolide. The specificity of the selected clone for triptolide was confirmed by ELISA and immunoprecipi-tation. After DNA sequencing, a BLAST search was carried out and 77 matching sequences was retrieved. The most promising candidate is human steroidogenic factor-1 (hSF-1), a member of the nuclear receptor family that controls the synthesis of steroid hormones by regulating steroidogenic enzyme genes. We purified the ligand binding domain (LBD) of hSF-1 and then used it for further experiments. Fluorescence spectrum experiments showed that triptolide could cause fluorescence quenching of hSF-1-LBD, isothermal titration calorimetric(ITC) measurements showed a enthalpydriven interaction between triptolide and hSF-1-LBD, and a dose-dependent interaction between them was observed by surface plasmon resonance(SPR). All these results confirmed the specific interaction between hSF-1 and triptolide.%采用噬菌体展示肽库技术筛选雷公藤内酯醇的靶蛋白,得到了一个肽段,并通过酶联免疫吸附(ELISA)和免疫共沉淀验证了该片段对雷公藤内酯醇的结合特异性.用Basic Local Alignment Search Tool (BLAST)进行序列对比后,找到了77个匹配序列,其中最为匹配的序列是人类类固醇生成因子-1(hSF-1),因此hSF-1可能是雷公藤内酯醇的一个潜在受体.在大肠杆菌中表达纯化了hSF-1的配体结合域(LBD),荧光光谱实验表明雷公藤内酯醇对hSF-1-LBD有荧光淬灭作用、等温滴定量热(ITC)实验表明雷公藤内酯醇与hSF-1-LBD发生焓驱动的特异性结合,表面等离子体共振(SPR)实验表明雷公藤内酯醇可以与hSF-1-LBD剂量依赖性结合,这些都证实了hSF-1与雷公藤内酯醇存在特异性相互作用.

  7. Development of RI-based real-time display technology of apoptotic cell death

    Energy Technology Data Exchange (ETDEWEB)

    Park, Sang Hyun; Jagn, Beom Su; Hayu, Tyas Utami

    2012-01-15

    Apoptosis, or the programmed cell death, is the generally normal death of a cell in living organisms. Inappropriate apoptosis (either too little or too much) is a factor in many human disease including neurodegenerative diseases, autoimmune disorders and many types of cancer. Therefore, it is one of the most challenging and widely studied topics currently. Development of RI-based real-time display technology of apoptosis can be provided invaluable analysis data for diagnosis and treatment of various diseases. In this study, bifunctional chelator (BFC) for Tc-99m tricarbonyl was synthesized for ML-10 derivative radiolabeling. The formation of complexation of apoptotic cells was developed by combining the ML-10 moiety with the BFC for {sup 99m}Tc-tricarbonyl precursor. The results of this project will be utilized for the development of RI-Biomics Center-based Total Analysis System (TAS) through the optimization of equipment in the RI-Biomics Center.

  8. Identification of systemic lupus erythematosus infection-associated epitope by random 7 peptide libraries displayed on phage%SLE患者血清中病原体相关噬菌体7肽的检测

    Institute of Scientific and Technical Information of China (English)

    王垚; 张凤民; 王亚贤; 颜培宇; 于晓红; 蔡文辉; 李玉军; 胡云龙; 翟爱霞; 陈杨

    2011-01-01

    目的用噬菌体7肽库筛选系统性红斑狼疮(SLE)患者血清特异性抗体,测序分析其实际意义。方法先用30例正常人混合血清与噬菌体7肽库反应,未与正常人白清结合的7肽再与30例SLE患者混合血清结合,获得SLE血清特异性结合的噬菌体克隆。用患者混合血清进行Dot-ELISA实验鉴定获得的噬菌体克隆,进一步用SLE患者及正常人血清各12例筛选阳性噬菌体的混合克隆,确定阳性噬菌体克隆与个体血清之间的结合情况,并对最终鉴定的噬菌体克隆进行测序与比对分析。结果混合的阳性噬菌体克隆与SLE患者个体血清反应阳性率明显高于其与正常人血清的反应率;序列分析显示阳性噬菌体克隆的抗原表位与杆菌、球菌、弧菌等微生物有同源性,与裂殖酵母属、链球菌属、立克次(氏)体属等有100%同源性,与人类抗原表位无关。结论SLE患者血清中存在与病原体抗原表位结合的抗体成分,提示SLE可能与病原体感染有关。%Objective To screen and identify the phage expressing random 7 amino acid peptide specific to the systemic lupus erythematosus(SLE)and analyze its practical significance. Methods Using the phage random 7 peptide library screening, the SLE specific phage clones were obtained by mixing with the sera from 30 SLE patients' and 30 normal controls respectively. Dot-ELISA was used to identify the SLE specific phage clones reacting to the sera of SLE patients and normal controls. Finally, the identified phages expressing random 7 amino acid peptides were sequenced and analyzed. Results Total 12 phages expressing random 7 amino acid peptides were obtained by phage peptide library screening and dot-ELISA identification. Sequence analysis showed that the no homology was found between the sequences of the deduced amino acid of the 7 antigen peptides and the sequence of human antigens. Peptides homology was found with coccus, coli, and

  9. Optogenetic Stimulation of Peripheral Vagus Nerves using Flexible OLED Display Technology to Treat Chronic Inflammatory Disease and Mental Health Disorders

    Science.gov (United States)

    2016-03-31

    and veterans, including arthritis, systemic inflammatory response syndrome, inflammatory bowel disease, post -traumatic stress disorder (PTSD...provide the desired specificity and minimize unwanted physiological side effects during treatment. Low Cost Mass Production Technology The notional...transcutaneous VNS OLED ‘bandage’ would be manufactured on a thin plastic substrate using commercial thin- film , flexible-display technology

  10. Dual paraboloid reflector technology development and the commercialization for projection display

    Science.gov (United States)

    Li, Kenneth

    2007-02-01

    One major issue in microdisplay-based projection television has always been the need for more light. Without sufficient amount of light, the screen gain is usually the final parameter that is adjusted to produce acceptable on-axis brightness. As a result, one major disadvantage of rear projection television (RPTV) is narrow viewing angle when compared with other display technologies. Traditional reflector systems, including elliptical and parabolic reflectors, perform well in most cases, but are inefficient for smaller etendue values corresponding to smaller image panels. The common remedy is to make lamps with shorter and shorter arcs to increase the coupling efficiency, but the corresponding lifetime of the lamps are reduced and most of the time, these short arc lamps can only operate at low power, thus limiting the total output of the illuminating system. The Dual Paraboloid reflector (DPR) technology allows the use of higher power lamps while maintaining long life, thus allows wider viewing angles to be achieved. In addition, low power lamps when used with the DPR system allows such a long life operation that lamp replacement is not needed, allowing the RPTV to compete effectively with LCDTV and PDP systems. These advantages are also applicable to front projectors with long life lamps that lower ownership costs.

  11. Teaching systems thinking to 4th and 5th graders using Environmental Dashboard display technology.

    Science.gov (United States)

    Clark, Shane; Petersen, John E; Frantz, Cindy M; Roose, Deborah; Ginn, Joel; Rosenberg Daneri, Daniel

    2017-01-01

    Tackling complex environmental challenges requires the capacity to understand how relationships and interactions between parts result in dynamic behavior of whole systems. There has been convincing research that these "systems thinking" skills can be learned. However, there is little research on methods for teaching these skills to children or assessing their impact. The Environmental Dashboard is a technology that uses "sociotechnical" feedback-information feedback designed to affect thought and behavior. Environmental Dashboard (ED) combines real-time information on community resource use with images and words that reflect pro-environmental actions of community members. Prior research indicates that ED supports the development of systems thinking in adults. To assess its impact on children, the technology was installed in a primary school and children were passively exposed to ED displays. This resulted in no measurable impact on systems thinking skills. The next stage of this research examined the impact of actively integrating ED into lessons on electricity in 4th and 5th grade. This active integration enhanced both content-related systems thinking skills and content retention.

  12. Phage therapy in the food industry.

    Science.gov (United States)

    Endersen, Lorraine; O'Mahony, Jim; Hill, Colin; Ross, R Paul; McAuliffe, Olivia; Coffey, Aidan

    2014-01-01

    Despite advances in modern technologies, the food industry is continuously challenged with the threat of microbial contamination. The overuse of antibiotics has further escalated this problem, resulting in the increasing emergence of antibiotic-resistant foodborne pathogens. Efforts to develop new methods for controlling microbial contamination in food and the food processing environment are extremely important. Accordingly, bacteriophages (phages) and their derivatives have emerged as novel, viable, and safe options for the prevention, treatment, and/or eradication of these contaminants in a range of foods and food processing environments. Whole phages, modified phages, and their derivatives are discussed in terms of current uses and future potential as antimicrobials in the traditional farm-to-fork context, encompassing areas such as primary production, postharvest processing, biosanitation, and biodetection. The review also presents some safety concerns to ensure safe and effective exploitation of bacteriophages in the future.

  13. Investigating effective support mounts for fans during display by exploring new technologies

    Directory of Open Access Journals (Sweden)

    Sarah Bright

    2012-06-01

    Full Text Available Blaise Castle House Museum in Bristol (UK houses a large and impressive collection of 130 European fans ranging from 1700-1950 with 60 brisé and 70 other fans in a generally good condition.  This research project explored how to best support this sort of collection by using 3D fabrication technology to create bespoke mounts.  At present making mounts for fans is complex and time consuming due to the layering and structure of fans, and the need for accuracy if the mount is to support the fan sufficiently.  This project explored how creating a digital 3D image of a fan could enable 3D fabrication technologies to be used to produce an exact ‘imprint’ of the object ensuring that all areas were supported, allowing a more suitable fan display to be created.  This paper outlines the process of exploring the principles of creating mounts for fans; narrates the process of selecting suitable objects for display, and the process of creating a digitally-produced fan mount.Blaise Castle House Museum à Bristol (Royaume-Uni abrite une importante et impressionnante collection de 130 éventails européens, datés de 1700 à 1950, dont 60 brisés et 70 autres dans un bon état général. Ce projet de recherche consiste à présenter au mieux ce type de collection en utilisant la technologie de fabrication 3D pour créer des supports sur mesure. À l'heure actuelle la conception de présentoirs est complexe et prend du temps, en raison de la structure des objets, et d’un besoin extrême de précision lorsque l’objectif est d’apporter un soutien aux différentes composantes des éventails. Ce projet explore donc comment la création d'une image numérique 3D d'un éventail pourrait permettre à des technologies de fabrication en 3D d’être utilisées pour produire une exacte «empreinte» de l'objet et veiller à ce que toutes les composantes soient supportées, permettant ainsi de créer le présentoir. Ce document décrit le processus d

  14. Overview on Glasses-Free 3D Display Technologies%裸视3D显示技术概述

    Institute of Scientific and Technical Information of China (English)

    王琼华; 王爱红; 粱栋; 邓欢

    2011-01-01

    The viewers can perceive stereoscopic effect from glasses-free 3D(three-dimensioanl) displays without wearing any special aided devices such as glasses. Several technologies of glasses-free 3D displays have been developed as people's understanding about 3D displays deepens. This paper reviews the principle and properties of the main glasses-free 3D displays, including parallax barrier or lenticular lens based 3D display, integral imaging 3D display, volumetric 3D display and holography 3D display.%裸视三维(3D)显示中,观看者无需配戴眼镜等任何助视设备就能观看到立体效果.随着人们对3D显示的认识不断加深,已提出多种裸视3D显示技术.本文综述了目前主流的裸视3D显示技术,包括光栅3D显示、集成成像3D显示、体3D显示和全息3D显示的基本原理及特性.

  15. Phage as a Genetically Modifiable Supramacromolecule in Chemistry, Materials and Medicine

    OpenAIRE

    Cao, Binrui; Yang, Mingying; Mao, Chuanbin

    2016-01-01

    Filamentous bacteriophage (phage) is a genetically modifiable supramacromolecule. It can be pictured as a semiflexible nanofiber (~900 nm long and ~8 nm wide) made of a DNA core and a protein shell with the former genetically encoding the latter. Although phage bioengineering and phage display techniques were developed before the 1990s, these techniques have not been widely used for chemistry, materials, and biomedical research from the perspective of supramolecular chemistry until recently. ...

  16. Novel Variants of Streptococcus thermophilus Bacteriophages Are Indicative of Genetic Recombination among Phages from Different Bacterial Species

    Science.gov (United States)

    Szymczak, Paula; Neves, Ana Rute; Kot, Witold; Hansen, Lars H.; Lametsch, René; Neve, Horst; Franz, Charles M. A. P.

    2016-01-01

    ABSTRACT Bacteriophages are the main cause of fermentation failures in dairy plants. The majority of Streptococcus thermophilus phages can be divided into either cos- or pac-type phages and are additionally characterized by examining the V2 region of their antireceptors. We screened a large number of S. thermophilus phages from the Chr. Hansen A/S collection, using PCR specific for the cos- or pac-type phages, as well as for the V2 antireceptor region. Three phages did not produce positive results with the assays. Analysis of phage morphologies indicated that two of these phages, CHPC577 and CHPC926, had shorter tails than the traditional S. thermophilus phages. The third phage, CHPC1151, had a tail size similar to those of the cos- or pac-type phages, but it displayed a different baseplate structure. Sequencing analysis revealed the genetic similarity of CHPC577 and CHPC926 with a subgroup of Lactococcus lactis P335 phages. Phage CHPC1151 was closely related to the atypical S. thermophilus phage 5093, homologous with a nondairy streptococcal prophage. By testing adsorption of the related streptococcal and lactococcal phages to the surface of S. thermophilus and L. lactis strains, we revealed the possibility of cross-interactions. Our data indicated that the use of S. thermophilus together with L. lactis, extensively applied for dairy fermentations, triggered the recombination between phages infecting different bacterial species. A notable diversity among S. thermophilus phage populations requires that a new classification of the group be proposed. IMPORTANCE Streptococcus thermophilus is a component of thermophilic starter cultures commonly used for cheese and yogurt production. Characterizing streptococcal phages, understanding their genetic relationships, and studying their interactions with various hosts are the necessary steps for preventing and controlling phage attacks that occur during dairy fermentations. PMID:28039135

  17. Phage “delay” towards enhancing bacterial escape from biofilms: a more comprehensive way of viewing resistance to bacteriophages

    Directory of Open Access Journals (Sweden)

    Stephen T. Abedon

    2017-03-01

    Full Text Available In exploring bacterial resistance to bacteriophages, emphasis typically is placed on those mechanisms which completely prevent phage replication. Such resistance can be detected as extensive reductions in phage ability to form plaques, that is, reduced efficiency of plating. Mechanisms include restriction-modification systems, CRISPR/Cas systems, and abortive infection systems. Alternatively, phages may be reduced in their “vigor” when infecting certain bacterial hosts, that is, with phages displaying smaller burst sizes or extended latent periods rather than being outright inactivated. It is well known, as well, that most phages poorly infect bacteria that are less metabolically active. Extracellular polymers such as biofilm matrix material also may at least slow phage penetration to bacterial surfaces. Here I suggest that such “less-robust” mechanisms of resistance to bacteriophages could serve bacteria by slowing phage propagation within bacterial biofilms, that is, delaying phage impact on multiple bacteria rather than necessarily outright preventing such impact. Related bacteria, ones that are relatively near to infected bacteria, e.g., roughly 10+ µm away, consequently may be able to escape from biofilms with greater likelihood via standard dissemination-initiating mechanisms including erosion from biofilm surfaces or seeding dispersal/central hollowing. That is, given localized areas of phage infection, so long as phage spread can be reduced in rate from initial points of contact with susceptible bacteria, then bacterial survival may be enhanced due to bacteria metaphorically “running away” to more phage-free locations. Delay mechanisms—to the extent that they are less specific in terms of what phages are targeted—collectively could represent broader bacterial strategies of phage resistance versus outright phage killing, the latter especially as require specific, evolved molecular recognition of phage presence. The

  18. Diversity of phage infection types and associated terminology: the problem with 'Lytic or lysogenic'.

    Science.gov (United States)

    Hobbs, Zack; Abedon, Stephen T

    2016-04-01

    Bacteriophages, or phages, are viruses of members of domain Bacteria. These viruses play numerous roles in shaping the diversity of microbial communities, with impact differing depending on what infection strategies specific phages employ. From an applied perspective, these especially are communities containing undesired or pathogenic bacteria that can be modified through phage-mediated bacterial biocontrol, that is, through phage therapy. Here we seek to categorize phages in terms of their infection strategies as well as review or suggest more descriptive, accurate or distinguishing terminology. Categories can be differentiated in terms of (1) whether or not virion release occurs (productive infections versus lysogeny, pseudolysogeny and/or the phage carrier state), (2) the means of virion release (lytic versus chronic release) and (3) the degree to which phages are genetically equipped to display lysogenic cycles (temperate versus non-temperate phages). We address in particular the use or overuse of what can be a somewhat equivocal phrase, 'Lytic or lysogenic', especially when employed as a means of distinguishing among phages types. We suggest that the implied dichotomy is inconsistent with both modern as well as historical understanding of phage biology. We consider, therefore, less ambiguous terminology for distinguishing between 'Lytic' versus 'Lysogenic' phage types.

  19. Genetically manipulated phages with improved pH resistance for oral administration in veterinary medicine

    Science.gov (United States)

    Nobrega, Franklin L.; Costa, Ana Rita; Santos, José F.; Siliakus, Melvin F.; van Lent, Jan W. M.; Kengen, Servé W. M.; Azeredo, Joana; Kluskens, Leon D.

    2016-01-01

    Orally administered phages to control zoonotic pathogens face important challenges, mainly related to the hostile conditions found in the gastrointestinal tract (GIT). These include temperature, salinity and primarily pH, which is exceptionally low in certain compartments. Phage survival under these conditions can be jeopardized and undermine treatment. Strategies like encapsulation have been attempted with relative success, but are typically complex and require several optimization steps. Here we report a simple and efficient alternative, consisting in the genetic engineering of phages to display lipids on their surfaces. Escherichia coli phage T7 was used as a model and the E. coli PhoE signal peptide was genetically fused to its major capsid protein (10 A), enabling phospholipid attachment to the phage capsid. The presence of phospholipids on the mutant phages was confirmed by High Performance Thin Layer Chromatography, Dynamic Light Scattering and phospholipase assays. The stability of phages was analysed in simulated GIT conditions, demonstrating improved stability of the mutant phages with survival rates 102–107 pfu.mL−1 higher than wild-type phages. Our work demonstrates that phage engineering can be a good strategy to improve phage tolerance to GIT conditions, having promising application for oral administration in veterinary medicine. PMID:27976713

  20. EVALUATION OF DISTRIBUTION HISTOGRAMS FOR INCREMENT OF CHROMATICITY COORDINATES IN DISPLAY TECHNOLOGIES

    Directory of Open Access Journals (Sweden)

    I. O. Zharinov

    2016-05-01

    Full Text Available We consider evaluation problem of chromaticity coordinates increment for an image displayed by indicating means (liquid crystal panels and etc.. Display device profile set by the weight matrix for components of primary colors serves as basic data for quantitative calculation. Research results have the form of mathematical expressions allowing calculation of increment values of chromaticity coordinates of the image displayed on indicating means and histograms of increment distribution.

  1. 助视3D显示技术概述%Overview on Glasses-Based 3D Display Technologies

    Institute of Scientific and Technical Information of China (English)

    王琼华; 粱栋; 宋呈群

    2011-01-01

    助视3D显示是指需要借助于3D眼镜等助视设备才能观看到3D图像的显示方式,它包括分色3D显示、偏振光3D显示和液晶快门3D显示.助视3D显示是目前最成熟的3D显示方式,近几年其相关技术及器件的性能得到了改进和提高.本文将介绍这三种助视3D显示技术.%Glasses-based 3D display is a kind of 3D displays which need viewers to wear special glasses to obtain stereoscopic vision. It can be classified into color separation-based 3D display, polarization-based 3D display and liquid crystal shutter-based 3D display. Nowadays it is the most mature 3D display. During recent years, its technologies and devices have made progress. This paper introduces these three types of glasses-based 3D displays.

  2. Improved Fab presentation on phage surface with the use of molecular chaperone coplasmid system.

    Science.gov (United States)

    Loh, Qiuting; Leong, Siew Wen; Tye, Gee Jun; Choong, Yee Siew; Lim, Theam Soon

    2015-05-15

    The low presentation efficiency of Fab (fragment antigen binding) fragments during phage display is largely due to the complexity of disulphide bond formation. This can result in the presentation of Fab fragments devoid of a light chain during phage display. Here we propose the use of a coplasmid system encoding several molecular chaperones (DsbA, DsbC, FkpA, and SurA) to improve Fab packaging. A comparison was done using the Fab fragment from IgG and IgD. We found that the use of the coplasmid during phage packaging was able to improve the presentation efficiency of the Fab fragment on phage surfaces. A modified version of panning using the coplasmid system was evaluated and was successful at enriching Fab binders. Therefore, the coplasmid system would be an attractive alternative for improved Fab presentation for phage display.

  3. Projection displays

    Science.gov (United States)

    Chiu, George L.; Yang, Kei H.

    1998-08-01

    Projection display in today's market is dominated by cathode ray tubes (CRTs). Further progress in this mature CRT projector technology will be slow and evolutionary. Liquid crystal based projection displays have gained rapid acceptance in the business market. New technologies are being developed on several fronts: (1) active matrix built from polysilicon or single crystal silicon; (2) electro- optic materials using ferroelectric liquid crystal, polymer dispersed liquid crystals or other liquid crystal modes, (3) micromechanical-based transducers such as digital micromirror devices, and grating light valves, (4) high resolution displays to SXGA and beyond, and (5) high brightness. This article reviews the projection displays from a transducer technology perspective along with a discussion of markets and trends.

  4. Screening peptide mimotopes of blood group B carbohydrate antigen using phage display peptide library%随机十二肽噬菌体展示文库筛选血型B抗原模拟多肽的实验研究

    Institute of Scientific and Technical Information of China (English)

    李许锋; 罗敏; 邹建军; 岑东芝; 何克菲; 张积仁

    2011-01-01

    OBJECTIVE: To screen peptide mimotopes of blood group B carbohydrate antigen with high affinity for blood group B monoclonal antibody which can replace carbohydrate antigen using a phage display peptide library, and find a new tool for application of blood group B carbohydrate antigen. METHODS: A 12-mer phage peptide library was screened for 3 rounds by using a blood group B monoclonal antibody as target protein according to such a procedure as “adsorbing,eluting and amplification”, and positive clones were selected randomly,confirmed by sandwich ELISA, single strand DNA was extracted from these positive clones and sequenced, and the mimic peptides were deduced by the DNA sequence. RESULTS: After 3 rounds of effective bio-panning, two major mimic peptides with high affinity for target protein were obtained, one peptide sequence was TKNMLSLPVGPG,the other one was HSLKHTQMSYSS. CONCLUSION: The resuits shows that the motif identified through a 12- mer phage display peptide library can be mimiced and may be a substitute for blood group B antigen.%目的:筛选出替代血型B抗原的模拟多肽,用多肽抗原替代糖类抗原.方法:抗血型B抗原的单克隆抗体作为固相筛选靶分子,对随机十二肽噬菌体展示文库进行生物淘选(bio-panning),经包被-结合-洗脱-扩增等循环3轮,对筛选的克隆ELISA鉴定,并通过剂量依赖实验验证其结合特异性.最后提取DNA测序,确定模拟肽氨基酸序列.结果:3轮筛选结束,得到2个亲和力较强的十二肽序列TKNMLSL-PVGPG和HSLKHTQMSYSS.结论:经过生物筛选得到模拟多肽序列,利用噬菌体展示技术筛选糖类抗原的模拟肽具有可行性,为糖类抗原的研究提供一种新思路.

  5. Construction and selection of human Fab antibody phage display library of extracellular domain of HER 2%人源性抗HER2胞外段Fab噬菌体抗体库的构建及筛选

    Institute of Scientific and Technical Information of China (English)

    张为家; 刘孝荣; 李官成; 贺智敏

    2011-01-01

    目的:构建全人源抗HER2胞外段(HER7. ECD)噬菌体Fab抗体库,从中筛选出特异性的抗体,并对其进行鉴定.方法:体外致敏并用EB病毒(EBV)转化HER2高表达乳腺癌患者的外周血单核细胞(PBMC),用PCR分别扩增重链Fd和轻链k/λ基因.经Sac I/Xba I和Xho/SpeI双酶切,依次克隆入噬菌体载体pComb3中,并电转化大肠杆菌XL1Blue,以辅助噬菌体VCSM13进行超感染,构建抗HER2 ECD人源化Fab噬菌体抗体库.以纯化HER2 ECD蛋白为抗原进行3轮固相淘选,富集抗HER2 ECD的抗体,并随机挑选克隆进行ELISA,获得的阳性克隆进一步以Western blot鉴定其抗原结合活性,对其中活性最高的克隆进行DNA测序.结果:构建了容量为2. 5 X 107的抗HER2 ECD的Fab噬菌体抗体库,并筛选获得了4株与HE2 ECD特异性结合的阳性克隆,Western blot分析显示其与HER?-ECD能较好的结合.选取结合活性最高的阳性克隆进行DNA序列分析,结果显示其重链可变区、轻链可变区分别与人胚系免疫球蛋白基因有高度的同源性.结论:成功构建了全人源抗HER2 ECD噬菌体抗体库,并筛选出抗HER2 ECD特异性较强的噬菌体克隆,为获得新的有临床应用价值的HER2 ECD抗体提供了实验基础.%AIM: To construct the fully humanized antiextracellular domain (ECD) of HER2.Fab fragment phage library, select antibodies against HER2 ECD specifically and identify its characteristics.METHODS: Peripheral blood monouclear cells (PBMCs) of breast cancer patients with HER2-overexpressing were immunized in vitro with purification protein of recombinant HEF2 ECD and were then transformed by Epstein-Barr virus (EBV).After total RNA was extracted, the heavy chain Fd and k/λ light chain were am plified by RT-PCR.Following restrictive digestion with Sac I/ Xba I and Xho I/Spe I, the light chain κ/λ genes and heavy chain genes Fd were inserted into the phagemid vector pComb3 successively and then electroporated into E.coil XL1-Blue

  6. P1-7: Modern Display Technology in Vision Science: Assessment of OLED and LCD Monitors for Visual Experiments

    Directory of Open Access Journals (Sweden)

    Tobias Elze

    2012-10-01

    Full Text Available For many decades, cathode ray tube (CRT monitors have been the dominant display technology in vision science. However, in recent years, most manufacturers stopped their CRT production lines, which enforces the application of alternative display technology in the field of vision science. Here, we analyze liquid crystal displays (LCDs and organic light-emitting diode (OLED monitors for their applicability in vision science experiments. Based on extensive measurements of their photometric output, we compare these technologies and contrast them with classical CRT monitors. Vision scientists aim to accurately present both static and dynamic stimuli on their display devices. As for the presentation of static stimuli, we demonstrate an increased accuracy for LCD and OLED devices compared to CRT monitors, because the former exhibit a higher degree of independence of neighboring pixels. As for dynamic presentations, both CRTs and OLEDs outperform LCD devices in terms of accuracy, because dynamic presentations on LCDs require a reorientation of the liquid crystal molecules, so that successive frames in time depend on each other. Together with widely unknown and uncontrolled technical artifacts, these properties of LCDs may impair visual experiments that require high temporal precision. Therefore, OLED monitors are more suitable for vision science experiments with respect to both their static and their temporal characteristics. However, for certain applications in visual neuroscience, the low duty cycle of some OLED devices may introduce frequencies to the photometric output which fall within the window of visibility of neurons in the visual cortex and therefore interfere with single unit recordings.

  7. Clostridium difficile phages: still difficult?

    Directory of Open Access Journals (Sweden)

    Katherine Rose Hargreaves

    2014-04-01

    Full Text Available Phages that infect Clostridium difficile were first isolated for typing purposes in the 1980s, but their use was short lived. However, the rise of C. difficile epidemics over the last decade has triggered a resurgence of interest in using phages to combat this pathogen. Phage therapy is an attractive treatment option for C. difficile infection, however developing suitable phages is challenging. In this review we summarise the difficulties faced by researchers in this field, and we discuss the solutions and strategies used for the development of C. difficile phages for use as novel therapeutics.Epidemiological data has highlighted the diversity and distribution of C. difficile, and shown that novel strains continue to emerge in clinical settings. In parallel with epidemiological studies, advances in molecular biology have bolstered our understanding of C. difficile biology, and our knowledge of phage-host interactions in other bacterial species. These three fields of biology have therefore paved the way for future work on C. difficile phages to progress and develop. Benefits of using C. difficile phages as therapeutic agents include the fact that they have highly specific interactions with their bacterial hosts. Studies also show that they can reduce bacterial numbers in both in vitro and in vivo systems. Genetic analysis has revealed the genomic diversity among these phages and provided an insight into their taxonomy and evolution.No strictly virulent C. difficile phages have been reported and this contributes to the difficulties with their therapeutic exploitation. Although treatment approaches using the phage-encoded endolysin protein have been explored, the benefits of using whole-phages are such that they remain a major research focus. Whilst we don’t envisage working with C. difficile phages will be problem free, sufficient study should inform future strategies to facilitate their development to combat this problematic pathogen.

  8. PhageTerm: a tool for fast and accurate determination of phage termini and packaging mechanism using next-generation sequencing data.

    Science.gov (United States)

    Garneau, Julian R; Depardieu, Florence; Fortier, Louis-Charles; Bikard, David; Monot, Marc

    2017-08-15

    The worrying rise of antibiotic resistance in pathogenic bacteria is leading to a renewed interest in bacteriophages as a treatment option. Novel sequencing technologies enable description of an increasing number of phage genomes, a critical piece of information to understand their life cycle, phage-host interactions, and evolution. In this work, we demonstrate how it is possible to recover more information from sequencing data than just the phage genome. We developed a theoretical and statistical framework to determine DNA termini and phage packaging mechanisms using NGS data. Our method relies on the detection of biases in the number of reads, which are observable at natural DNA termini compared with the rest of the phage genome. We implemented our method with the creation of the software PhageTerm and validated it using a set of phages with well-established packaging mechanisms representative of the termini diversity, i.e. 5'cos (Lambda), 3'cos (HK97), pac (P1), headful without a pac site (T4), DTR (T7) and host fragment (Mu). In addition, we determined the termini of nine Clostridium difficile phages and six phages whose sequences were retrieved from the Sequence Read Archive. PhageTerm is freely available (https://sourceforge.net/projects/phageterm), as a Galaxy ToolShed and on a Galaxy-based server (https://galaxy.pasteur.fr).

  9. Phage neutralization by sera of patients receiving phage therapy.

    Science.gov (United States)

    Łusiak-Szelachowska, Marzanna; Zaczek, Maciej; Weber-Dąbrowska, Beata; Międzybrodzki, Ryszard; Kłak, Marlena; Fortuna, Wojciech; Letkiewicz, Sławomir; Rogóż, Paweł; Szufnarowski, Krzysztof; Jończyk-Matysiak, Ewa; Owczarek, Barbara; Górski, Andrzej

    2014-08-01

    The aim of our investigation was to verify whether phage therapy (PT) can induce antiphage antibodies. The antiphage activity was determined in sera from 122 patients from the Phage Therapy Unit in Wrocław with bacterial infections before and during PT, and in sera from 30 healthy volunteers using a neutralization test. Furthermore, levels of antiphage antibodies were investigated in sera of 19 patients receiving staphylococcal phages and sera of 20 healthy volunteers using enzyme-linked immunosorbent assay. The phages were administered orally, locally, orally/locally, intrarectally, or orally/intrarectally. The rate of phage inactivation (K) estimated the level of phages' neutralization by human sera. Low K rates were found in sera of healthy volunteers (K ≤ 1.73). Low K rates were detected before PT (K ≤ 1.64). High antiphage activity of sera K > 18 was observed in 12.3% of examined patients (n = 15) treated with phages locally (n = 13) or locally/orally (n = 2) from 15 to 60 days of PT. High K rates were found in patients treated with some Staphylococcus aureus, Pseudomonas aeruginosa, and Enterococcus faecalis phages. Low K rates were observed during PT in sera of patients using phages orally (K ≤ 1.04). Increased inactivation of phages by sera of patients receiving PT decreased after therapy. These results suggest that the antiphage activity in patients' sera depends on the route of phage administration and phage type. The induction of antiphage activity of sera during or after PT does not exclude a favorable result of PT.

  10. Characterization of Campylobacter phages including analysis of host range by selected Campylobacter Penner serotypes

    DEFF Research Database (Denmark)

    Hansen, Vinni; Rosenquist, Hanne; Baggesen, Dorte Lau

    2007-01-01

    Background: The predominant food borne pathogen in the western world today is Campylobacter. Campylobacter specific bacteriophages (phages) have been proposed as an alternative agent for reducing the burden of Campylobacter in broilers. One concern in relation to phage biocontrol is the narrow host...... range often displayed by phages. To identify the potential of phages as a Campylobacter reducing agent we needed to determine their infectivity on a panel of isolates representing the Campylobacter strains found in broilers as well as humans. Results: In this study, Campylobacter phages were isolated...... from the intestines of broilers and ducks and from abattoir sewage. Twelve phages were investigated to determine their ability to infect the Campylobacter Penner serotypes commonly present in Danish poultry and patients with campylobacteriosis. A total of 89% of the Campylobacter jejuni strains and 14...

  11. Phage as a Genetically Modifiable Supramacromolecule in Chemistry, Materials and Medicine.

    Science.gov (United States)

    Cao, Binrui; Yang, Mingying; Mao, Chuanbin

    2016-06-21

    Filamentous bacteriophage (phage) is a genetically modifiable supramacromolecule. It can be pictured as a semiflexible nanofiber (∼900 nm long and ∼8 nm wide) made of a DNA core and a protein shell with the former genetically encoding the latter. Although phage bioengineering and phage display techniques were developed before the 1990s, these techniques have not been widely used for chemistry, materials, and biomedical research from the perspective of supramolecular chemistry until recently. Powered by our expertise in displaying a foreign peptide on its surface through engineering phage DNA, we have employed phage to identify target-specific peptides, construct novel organic-inorganic nanohybrids, develop biomaterials for disease treatment, and generate bioanalytical methods for disease diagnosis. Compared with conventional biomimetic chemistry, phage-based supramolecular chemistry represents a new frontier in chemistry, materials science, and medicine. In this Account, we introduce our recent successful efforts in phage-based supramolecular chemistry, by integrating the unique nanofiber-like phage structure and powerful peptide display techniques into the fields of chemistry, materials science, and medicine: (1) successfully synthesized and assembled silica, hydroxyapatite, and gold nanoparticles using phage templates to form novel functional materials; (2) chemically introduced azo units onto the phage to form photoresponsive functional azo-phage nanofibers via a diazotization reaction between aromatic amino groups and the tyrosine residues genetically displayed on phage surfaces; (3) assembled phage into 2D films for studying the effects of both biochemical (the peptide sequences displayed on the phages) and biophysical (the topographies of the phage films) cues on the proliferation and differentiation of mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs) and identified peptides and topographies that can induce their osteogenic

  12. Relevant uses of surface proteins--display on self-organized biological structures.

    Science.gov (United States)

    Jahns, Anika C; Rehm, Bernd H A

    2012-03-01

    Proteins are often found attached to surfaces of self-assembling biological units such as whole microbial cells or subcellular structures, e.g. intracellular inclusions. In the last two decades surface proteins were identified that could serve as anchors for the display of foreign protein functions. Extensive protein engineering based on structure-function data enabled efficient display of technically and/or medically relevant protein functions. Small size, diversity of the anchor protein as well as support structure, genetic manipulability and controlled cultivation of phages, bacterial cells and yeasts contributed to the establishment of designed and specifically functionalized tools for applications as sensors, catalysis, biomedicine, vaccine development and library-based screening technologies. Traditionally, phage display is employed for library screening but applications in biomedicine and vaccine development are also perceived. For some diagnostic purposes phages are even too small in size so other carrier materials where needed and gave way for cell and yeast display. Only recently, intracellular inclusions such as magnetosomes, polyhydroxyalkanoate granules and lipid bodies were conceived as stable subcellular structures enabling the display of foreign protein functions and showing potential as specific and tailor-made devices for medical and biotechnological applications.

  13. Selection of gonadotrophin surge attenuating factor phage antibodies by bioassay

    Directory of Open Access Journals (Sweden)

    Mason Helen D

    2005-09-01

    bioassay and phage display technologies is a powerful tool in the study of uncharacterised proteins that defy conventional approaches. In addition, we conclude that these data support suggestions that GnSAF may be structurally related to serum albumin or very tightly bound to serum albumin.

  14. Hybrid Nanomaterial Complexes for Advanced Phage-guided Gene Delivery

    Directory of Open Access Journals (Sweden)

    Teerapong Yata

    2014-01-01

    Full Text Available Developing nanomaterials that are effective, safe, and selective for gene transfer applications is challenging. Bacteriophages (phage, viruses that infect bacteria only, have shown promise for targeted gene transfer applications. Unfortunately, limited progress has been achieved in improving their potential to overcome mammalian cellular barriers. We hypothesized that chemical modification of the bacteriophage capsid could be applied to improve targeted gene delivery by phage vectors into mammalian cells. Here, we introduce a novel hybrid system consisting of two classes of nanomaterial systems, cationic polymers and M13 bacteriophage virus particles genetically engineered to display a tumor-targeting ligand and carry a transgene cassette. We demonstrate that the phage complex with cationic polymers generates positively charged phage and large aggregates that show enhanced cell surface attachment, buffering capacity, and improved transgene expression while retaining cell type specificity. Moreover, phage/polymer complexes carrying a therapeutic gene achieve greater cancer cell killing than phage alone. This new class of hybrid nanomaterial platform can advance targeted gene delivery applications by bacteriophage.

  15. Technical solutions for a full-resolution autostereoscopic 2D/3D display technology

    Science.gov (United States)

    Stolle, Hagen; Olaya, Jean-Christophe; Buschbeck, Steffen; Sahm, Hagen; Schwerdtner, Armin

    2008-02-01

    Auto-stereoscopic 3D displays capable of high quality, full-resolution images for multiple users can only be created with time-sequential systems incorporating eye tracking and a dedicated optical design. The availability of high speed displays with 120Hz and faster eliminated one of the major hurdles for commercial solutions. Results of alternative display solutions from SeeReal show the impact of optical design on system performance and product features. Depending on the manufacturer's capabilities, system complexity can be shifted from optics to SLM with an impact on viewing angle, number of users and energy efficiency, but also on manufacturing processes. A proprietary solution for eye tracking from SeeReal demonstrates that the required key features can be achieved and implemented in commercial systems in a reasonably short time.

  16. An integrated impact assessment and weighting methodology: evaluation of the environmental consequences of computer display technology substitution.

    Science.gov (United States)

    Zhou, Xiaoying; Schoenung, Julie M

    2007-04-01

    Computer display technology is currently in a state of transition, as the traditional technology of cathode ray tubes is being replaced by liquid crystal display flat-panel technology. Technology substitution and process innovation require the evaluation of the trade-offs among environmental impact, cost, and engineering performance attributes. General impact assessment methodologies, decision analysis and management tools, and optimization methods commonly used in engineering cannot efficiently address the issues needed for such evaluation. The conventional Life Cycle Assessment (LCA) process often generates results that can be subject to multiple interpretations, although the advantages of the LCA concept and framework obtain wide recognition. In the present work, the LCA concept is integrated with Quality Function Deployment (QFD), a popular industrial quality management tool, which is used as the framework for the development of our integrated model. The problem of weighting is addressed by using pairwise comparison of stakeholder preferences. Thus, this paper presents a new integrated analytical approach, Integrated Industrial Ecology Function Deployment (I2-EFD), to assess the environmental behavior of alternative technologies in correlation with their performance and economic characteristics. Computer display technology is used as the case study to further develop our methodology through the modification and integration of various quality management tools (e.g., process mapping, prioritization matrix) and statistical methods (e.g., multi-attribute analysis, cluster analysis). Life cycle thinking provides the foundation for our methodology, as we utilize a published LCA report, which stopped at the characterization step, as our starting point. Further, we evaluate the validity and feasibility of our methodology by considering uncertainty and conducting sensitivity analysis.

  17. Epitope screening of influenza A (H3N2) by using phage display library%应用噬菌体表面展示技术筛选流感病毒H3N2抗原模拟表位

    Institute of Scientific and Technical Information of China (English)

    钟彦伟; 徐东平; 李晓东; 戴久增; 许彪; 李乐

    2009-01-01

    Objective To screen the influenza A (H3N2) mimotopes by using phage display library. Methods Using influenza A (H3N2) monaclonai antibody as selective molecule, a 7 mer phage peptide library was biopanned and positive clones were selected by ELISA, competition assay and DNA sequencing. Results 21 positive clones were chosen for DNA sequencing. From the experiment and sequencing comparison results, one epitope was comfirmed as mimotope of influenza A (H3N2). Conclusion Influenza A (H3N2) mimotope was obtained by phage peptide library screening. The result provide a new approach for new Influenza vitals vaccine development.%目的 筛选特异性流感病毒H3N2抗原模拟表位,为开展新的流感病毒疫苗研究探索新的途径.方法 应用噬菌体表面展示技术,以抗-H3N2的单克隆抗体作为固相筛选分子,对人工合成的噬菌体随机肽库进行5轮"吸附-洗脱-扩增"的筛选过程,在第5轮筛选后,随机挑取48个克隆,经噬菌体酶联免疫吸附法(ELISA)鉴定并进行交叉反应实验以及竞争抑制性实验,最后对所选克隆进行DNA序列分析,以确定H3N2抗原的模拟表位.结果 经噬菌体富集后,从随机筛选的48个克隆中得到21个阳性克隆,经ELISA鉴定及交叉反应实验、竞争抑制性实验后,确定氨基酸序列XTXPYXX为H3N2的模拟表位.结论 用噬菌体7肽库筛选得到H3N2的模拟表位,为开展用流感病毒模拟表位探索新的防治方法研究奠定了基础.

  18. A variety of human monoclonal antibodies against epidermal growth factor receptor isolated from a phage antibody library.

    Science.gov (United States)

    Kurosawa, Gene; Kondo, Mariko; Kurosawa, Yoshikazu

    2016-11-04

    When the technology for constructing human antibody (Ab) libraries using a phage-display system was developed, many researchers in Ab-related fields anticipated that it would be widely applied to the development of pharmaceutical drugs against various diseases, including cancers. However, successful examples of such applications are very limited. Moreover, researchers who utilize phage-display technology now show divergent ways of thinking about phage Ab libraries. For example, there is debate about what should be the source of VH and VL genes for the construction of libraries to cover the whole repertoire of Abs present in the human body. In the immune system, the introduction of mutations into V genes followed by selection based on binding activity, termed Ab maturation, is required for the production of Abs exhibiting high affinity to the antigen (Ag). Therefore, introduction of mutations and selection are required for isolation of Abs with high affinity after isolation of clones from phage Ab libraries. We constructed a large human Ab library termed AIMS, developed a screening method termed ICOS, and succeeded in isolating many human monoclonal Abs (mAbs) that specifically and strongly bind to various tumor-associated Ags. Eight anti-EGFR mAbs were included, which we characterized. These mAbs showed various different activities against EGFR-expressing cancer cells. In this paper, we describe these data and discuss the possibility and necessity that the mAbs isolated from the AIMS library might be developed as therapeutic drugs against cancers without introduction of mutations. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  19. Killing cancer cells by targeted drug-carrying phage nanomedicines

    Directory of Open Access Journals (Sweden)

    Yacoby Iftach

    2008-04-01

    Full Text Available Abstract Background Systemic administration of chemotherapeutic agents, in addition to its anti-tumor benefits, results in indiscriminate drug distribution and severe toxicity. This shortcoming may be overcome by targeted drug-carrying platforms that ferry the drug to the tumor site while limiting exposure to non-target tissues and organs. Results We present a new form of targeted anti-cancer therapy in the form of targeted drug-carrying phage nanoparticles. Our approach is based on genetically-modified and chemically manipulated filamentous bacteriophages. The genetic manipulation endows the phages with the ability to display a host-specificity-conferring ligand. The phages are loaded with a large payload of a cytotoxic drug by chemical conjugation. In the presented examples we used anti ErbB2 and anti ERGR antibodies as targeting moieties, the drug hygromycin conjugated to the phages by a covalent amide bond, or the drug doxorubicin conjugated to genetically-engineered cathepsin-B sites on the phage coat. We show that targeting of phage nanomedicines via specific antibodies to receptors on cancer cell membranes results in endocytosis, intracellular degradation, and drug release, resulting in growth inhibition of the target cells in vitro with a potentiation factor of >1000 over the corresponding free drugs. Conclusion The results of the proof-of concept study presented here reveal important features regarding the potential of filamentous phages to serve as drug-delivery platform, on the affect of drug solubility or hydrophobicity on the target specificity of the platform and on the effect of drug release mechanism on the potency of the platform. These results define targeted drug-carrying filamentous phage nanoparticles as a unique type of antibody-drug conjugates.

  20. Panning and identification of antagonistic active peptides specifically bin-ding to the first and second extracellular membrane loops of rat CCR5 by technique of phage display peptide library%应用噬菌体展示肽库技术淘选大鼠 CCR5膜外第一、二胞外环特异性结合的活性拮抗肽与初步鉴定

    Institute of Scientific and Technical Information of China (English)

    刘思雪; 胡梅; 叶小研; 黄花荣; 钟英强

    2015-01-01

    [ ABSTRACT] AIM: To pan the active peptides which specifically bound to the first and second extracellular membrane loops of rat CC chemokine receptor 5 ( CCR5 ) .METHODS: The technique of phage display peptide library was used and binding ability of the peptides was identified.The amino acid sequences of the first and second extracellular loops of rat CCR5 were searched in the protein database and chemically synthesized corresponding linear peptides were used as targets in the biopanning.After 3 to 4 rounds of screening with Ph.D.TM-7 Phage Display Peptide Library were per-formed, the specific phages were collected and primarily identified by ELISA.RESULTS:The sequences of the peptides displayed on the selected phages were GHWKVWL and HYIDFRW, both of them exhibited positive in phage binding ELISA and the binding to phages and targets were concentration dependent and saturable.CONCLUSION:Two antagonis-tic active peptides specifically binding to CCR5 were successfully obtained by the technique of phage display peptide librar-y, and the binding ability to the first and second extracellular membrane loops of rat CCR5 were proved in vitro.%目的:利用噬菌体展示肽库技术淘选与大鼠CC趋化因子受体5( CCR5)膜外第一、二胞外环特异性结合的短肽,并鉴定其与CCR5的结合能力。方法:在蛋白质数据库中查得大鼠CCR5第一、二胞外环的氨基酸序列,合成相应的线性短肽作为淘选的靶分子,利用噬菌体展示7肽文库进行3~4轮淘选,用ELISA法鉴定所选肽与靶分子的结合,并测定其与浓度的关系。结果:与CCR5第一、二胞外环特异性结合的噬菌体展示的短肽序列分别为GHWKVWL和HYIDFRW,ELISA鉴定呈阳性反应,且短肽与靶分子的结合具有浓度依赖性和可饱和性。结论:利用噬菌体展示技术成功获得了2条CCR5特异性结合的短肽,并在体外证明其可与CCR5第一、二胞外环具有结合能力。

  1. Enhancement display of veins distribution based on binocular vision and image fusion technology

    Science.gov (United States)

    Liu, Peng; Di, Si; Jin, Jian; Bai, Liping

    2014-11-01

    The capture and display of veins distribution is an important issue for some applications, such as medical diagnosis and identification. Therefore, it has become a popular topic in the field of biomedical imaging. Usually, people capture the veins distribution by infrared imaging, but the display result is similar with that of a gray picture and the color and details of skin cannot be remained. To some degree, it is unreal for doctors. In this paper, we develop a binocular vision system to carry out the enhancement display of veins under the condition of keeping actual skin color. The binocular system is consisted of two adjacent cameras. A visible band filter and an infrared band filter are placed in front of the two lenses, respectively. Therefore, the pictures of visible band and infrared band can be captured simultaneously. After that, a new fusion process is applied to the two pictures, which related to histogram mapping, principal component analysis (PCA) and modified bilateral filter fusion. The final results show that both the veins distribution and the actual skin color of the back of the hand can be clearly displayed. Besides, correlation coefficient, average gradient and average distortion are selected as the parameters to evaluate the image quality. By comparing the parameters, it is evident that our novel fusion method is prior to some popular fusion methods such as Gauss filter fusion, Intensity-hue-saturation (HIS) fusion and bilateral filter fusion.

  2. Helmet-mounted display technology on the VISTA NF-16D

    Science.gov (United States)

    Underhill, Gregory P.; Bailey, Randall E.; Markman, Steve

    1997-06-01

    Wright Laboratory's Variable-Stability In-Flight Simulator Test Aircraft (VISTA) NF-16D is the newest in-flight simulator in the USAF inventory. A unique research aircraft, it will perform a multitude of missions: to develop and evaluate flight characteristics of new aircraft that have not yet flown, and perform research in the areas of flying qualities, flight control design, pilot-vehicle interface, weapons and avionics integration, and to train new test pilots. The VISTA upgrade will enhance the simulation fidelity and research capabilities by adding a programmable helmet-mounted display (HMD) and head-up display (HUD) in the front cockpit. The programmable HMD consists of a GEC- Marconi Avionics Viper II Helmet-Mounted Optics Module integrated with a modified Helmet Integrated Systems Limited HGU-86/P helmet, the Honeywell Advanced Metal Tolerant tracker, and a GEC-Mounted Tolerant tracker, and a GEC- Marconi Avionics Programmable Display Generator. This system will provide a real-time programmable HUD and monocular stroke capable HMD in the front cockpit. The HMD system is designed for growth to stroke-on-video, binocular capability. This paper examines some of issues associated with current HMD development, and explains the value of rapid prototyping or 'quick-look' flight testing on the VISTA NF-16D. A brief overview of the VISTA NF-16D and the hardware and software modifications made to incorporate the programmable display system is give, as well as a review of several key decisions that were made in the programmable display system implementation. The system's capabilities and what they mean to potential users and designers are presented, particularly for pilot-vehicle interface research.

  3. Phage cocktails and the future of phage therapy.

    Science.gov (United States)

    Chan, Benjamin K; Abedon, Stephen T; Loc-Carrillo, Catherine

    2013-06-01

    Viruses of bacteria, known as bacteriophages or phages, were discovered nearly 100 years ago. Their potential as antibacterial agents was appreciated almost immediately, with the first 'phage therapy' trials predating Fleming's discovery of penicillin by approximately a decade. In this review, we consider phage therapy that can be used for treating bacterial infections in humans, domestic animals and even biocontrol in foods. Following an overview of the topic, we explore the common practice - both experimental and, in certain regions of the world, clinical - of mixing therapeutic phages into cocktails consisting of multiple virus types. We conclude with a discussion of the commercial and medical context of phage cocktails as therapeutic agents. In comparing off-the-shelf versus custom approaches, we consider the merits of a middle ground, which we deem 'modifiable'. Finally, we explore a regulatory framework for such an approach based on an influenza vaccine model.

  4. Enhanced/Synthetic Vision and Head-Worn Display Technologies for Terminal Maneuvering Area NextGen Operations

    Science.gov (United States)

    Arthur, Jarvis J., III; Prinzell, Lawrence J.; Williams, Steven P.; Bailey, Randall E.; Shelton, Kevin J.; Norman, R. Mike

    2011-01-01

    NASA is researching innovative technologies for the Next Generation Air Transportation System (NextGen) to provide a "Better-Than-Visual" (BTV) capability as adjunct to "Equivalent Visual Operations" (EVO); that is, airport throughputs equivalent to that normally achieved during Visual Flight Rules (VFR) operations rates with equivalent and better safety in all weather and visibility conditions including Instrument Meteorological Conditions (IMC). These new technologies build on proven flight deck systems and leverage synthetic and enhanced vision systems. Two piloted simulation studies were conducted to access the use of a Head-Worn Display (HWD) with head tracking for synthetic and enhanced vision systems concepts. The first experiment evaluated the use a HWD for equivalent visual operations to San Francisco International Airport (airport identifier: KSFO) compared to a visual concept and a head-down display concept. A second experiment evaluated symbology variations under different visibility conditions using a HWD during taxi operations at Chicago O'Hare airport (airport identifier: KORD). Two experiments were conducted, one in a simulated San Francisco airport (KSFO) approach operation and the other, in simulated Chicago O'Hare surface operations, evaluating enhanced/synthetic vision and head-worn display technologies for NextGen operations. While flying a closely-spaced parallel approach to KSFO, pilots rated the HWD, under low-visibility conditions, equivalent to the out-the-window condition, under unlimited visibility, in terms of situational awareness (SA) and mental workload compared to a head-down enhanced vision system. There were no differences between the 3 display concepts in terms of traffic spacing and distance and the pilot decision-making to land or go-around. For the KORD experiment, the visibility condition was not a factor in pilot's rating of clutter effects from symbology. Several concepts for enhanced implementations of an unlimited field

  5. ATM Technology Demonstration-1 Phase II Boeing Configurable Graphical Display (CGD) Software Design Description

    Science.gov (United States)

    Wilber, George F.

    2017-01-01

    This Software Description Document (SDD) captures the design for developing the Flight Interval Management (FIM) system Configurable Graphics Display (CGD) software. Specifically this SDD describes aspects of the Boeing CGD software and the surrounding context and interfaces. It does not describe the Honeywell components of the CGD system. The SDD provides the system overview, architectural design, and detailed design with all the necessary information to implement the Boeing components of the CGD software and integrate them into the CGD subsystem within the larger FIM system. Overall system and CGD system-level requirements are derived from the CGD SRS (in turn derived from the Boeing System Requirements Design Document (SRDD)). Display and look-and-feel requirements are derived from Human Machine Interface (HMI) design documents and working group recommendations. This Boeing CGD SDD is required to support the upcoming Critical Design Review (CDR).

  6. The epic of phage therapy

    OpenAIRE

    Alain Dublanchet; Shawna Bourne

    2007-01-01

    The present report describes the presentation given by Dr Alain Dublanchet at the Stanier/Oxford Hygiene Symposium, held in Oxford, England, on November 10, 2004. Dr Dublanchet's lecture, entitled ‘The epic of phage therapy’, provided a sequential account of the use of phage as an antimicrobial from its discovery to its rise and fall and current rediscovery.

  7. The influence of desk and display design on posture and muscle activity variability whilst performing information technology tasks.

    Science.gov (United States)

    Straker, L; Burgess-Limerick, R; Pollock, C; Maslen, B

    2009-09-01

    Desk design and computer display height can affect posture and muscle activation during computer use. Amplitudes of postural variables and muscle activity during computer use do not explain the results from epidemiological studies of musculoskeletal discomfort and disorders related to computer use. The purpose of this study was to assess variability of posture and muscle activity during work with two computer display heights and book/paper, in conjunction with a curved desk designed to provide forearm support and a traditional, straight desk. 18 male and 18 female participants performed 10-min tasks involving keying, mousing, reading and writing in six desk/display conditions. 3D posture and surface emg were assessed for the final 2 min of each task. The curved desk resulted in greater postural and muscle activity variation, suggesting an advantage of this supportive surface over the straight desk. There was little difference in variability associated with the two display heights. However, greater variability of posture and muscle activity was evident with the book/paper condition. Non-touch typists had greater neck flexion variation. The design of information technology tasks and workstations can influence the short term variation in posture and muscle activity. Variation is influenced independently of mean postures and muscle amplitudes and therefore needs to be considered to adequately assess the risk of musculoskeletal disorders.

  8. All solution processed organic thin film transistor-backplane with printing technology for electrophoretic display

    Science.gov (United States)

    Lee, Myung W.; Song, C.K.

    2012-01-01

    In this study, solution processes were developed for backplane using an organic thin film transistor (OTFT) as a driving device for an electrophoretic display (EPD) panel. The processes covered not only the key device of OTFTs but also interlayer and pixel electrodes. The various materials and printing processes were adopted to achieve the requirements of devices and functioning layers. The performance of OTFT of the backplane was sufficient to drive EPD sheet by producing a mobility of 0.12 cm2/v x sec and on/off current ratio of 10(5).

  9. Exploiting bacterial peptide display technology to engineer biomaterials for neural stem cell culture.

    Science.gov (United States)

    Little, Lauren E; Dane, Karen Y; Daugherty, Patrick S; Healy, Kevin E; Schaffer, David V

    2011-02-01

    Stem cells are often cultured on substrates that present extracellular matrix (ECM) proteins; however, the heterogeneous and poorly defined nature of ECM proteins presents challenges both for basic biological investigation of cell-matrix investigations and translational applications of stem cells. Therefore, fully synthetic, defined materials conjugated with bioactive ligands, such as adhesive peptides, are preferable for stem cell biology and engineering. However, identifying novel ligands that engage cellular receptors can be challenging, and we have thus developed a high throughput approach to identify new adhesive ligands. We selected an unbiased bacterial peptide display library for the ability to bind adult neural stem cells (NSCs), and 44 bacterial clones expressing peptides were identified and found to bind to NSCs with high avidity. Of these clones, four contained RGD motifs commonly found in integrin binding domains, and three exhibited homology to ECM proteins. Three peptide clones were chosen for further analysis, and their synthetic analogs were adsorbed on tissue culture polystyrene (TCPS) or grafted onto an interpenetrating polymer network (IPN) for cell culture. These three peptides were found to support neural stem cell self-renewal in defined medium as well as multi-lineage differentiation. Therefore, bacterial peptide display offers unique advantages to isolate bioactive peptides from large, unbiased libraries for applications in biomaterials engineering.

  10. Lambda-Display: A Powerful Tool for Antigen Discovery

    Directory of Open Access Journals (Sweden)

    Nicola Gargano

    2011-04-01

    Full Text Available Since its introduction in 1985, phage display technology has been successfully used in projects aimed at deciphering biological processes and isolating molecules of practical value in several applications. Bacteriophage lambda, representing a classical molecular cloning and expression system has also been exploited for generating large combinatorial libraries of small peptides and protein domains exposed on its capsid. More recently, lambda display has been consistently and successfully employed for domain mapping, antigen discovery and protein interaction studies or, more generally, in functional genomics. We show here the results obtained by the use of large libraries of cDNA and genomic DNA for the molecular dissection of the human B-cell response against complex pathogens, including protozoan parasites, bacteria and viruses. Moreover, by reviewing the experimental work performed in recent investigations we illustrate the potential of lambda display in the diagnostics field and for identifying antigens useful as targets for vaccine development.

  11. Phages targeting infected tissues: novel approach to phage therapy.

    Science.gov (United States)

    Górski, Andrzej; Dąbrowska, Krystyna; Hodyra-Stefaniak, Katarzyna; Borysowski, Jan; Międzybrodzki, Ryszard; Weber-Dąbrowska, Beata

    2015-01-01

    While the true efficacy of phage therapy still requires formal confirmation in clinical trials, it continues to offer realistic potential treatment in patients in whom antibiotics have failed. Novel developments and approaches are therefore needed to ascertain that future clinical trials would evaluate the therapy in its optimal form thus allowing for reliable conclusions regarding the true value of phage therapy. In this article, we present our vision to develop and establish a bank of phages specific to most threatening pathogens and armed with homing peptides enabling their localization in infected tissues in densities assuring efficient and stable eradication of infection.

  12. Radiation visualization in virtual reality: A comparison of flat and topographic map types, presented on four different display technologies

    Energy Technology Data Exchange (ETDEWEB)

    Nystad, Espen; Sebok, Angelia

    2005-08-15

    HWR-734 describes an experiment performed to compare different types of VR display technologies and their effects on learning. In the study, two different ways of presenting radiation information were compared. One was a flat radiation map with different colours for different levels of radiation. The other was a topographic map, where radiation levels were distinguished both by colour and by the elevation of the map. The efficiency of the maps for learning radiation information, and subjective preferences was assessed. The results indicated that the maps were each suited for different kinds of use. It is recommended to follow up this study with further investigation of radiation map efficiency. (Author)

  13. Three-Dimensional Display Technologies for Anatomical Education: A Literature Review

    Science.gov (United States)

    Hackett, Matthew; Proctor, Michael

    2016-01-01

    Anatomy is a foundational component of biological sciences and medical education and is important for a variety of clinical tasks. To augment current curriculum and improve students' spatial knowledge of anatomy, many educators, anatomists, and researchers use three-dimensional (3D) visualization technologies. This article reviews 3D display…

  14. Flexible barrier technology for enabling rollable AMOLED displays and upscaling flexible OLED lighting

    NARCIS (Netherlands)

    Li, F.M.; Unnikrishnan, S.; Weijer, P. van de; Assche, F. van; Shen, J.; Ellis, T.; Manders, W.; Akkerman, H.; Bouten, P.; Mol, A.M.B. van

    2013-01-01

    The availability of a high performance thin-film barrier is the most critical challenge in upscaling and commercializing flexible OLED products. We report a flexible thin-film-barrier technology that meets lifetime specifications for OLED lighting, and demonstrate it in rollable QVGA a-IGZO AMOLED

  15. Leishmania infantum mimotopes and a phage-ELISA assay as tools for a sensitive and specific serodiagnosis of human visceral leishmaniasis.

    Science.gov (United States)

    Salles, Beatriz C S; Costa, Lourena E; Alves, Patrícia T; Dias, Ana C S; Vaz, Emília R; Menezes-Souza, Daniel; Ramos, Fernanda F; Duarte, Mariana C; Roatt, Bruno M; Chávez-Fumagalli, Miguel A; Tavares, Carlos A P; Gonçalves, Denise U; Rocha, Regina L; Goulart, Luiz R; Coelho, Eduardo A F

    2017-03-01

    Serological methods used to diagnose visceral leishmaniasis (VL) are considered minimally invasive, but they present problems related with their sensitivity and/or specificity. In this study, a subtractive selection using the phage display technology against antibodies from healthy subjects living in endemic and non-endemic areas of disease, as well as from Chagas disease patients and those developing active VL, was developed. The aim of this study was to select bacteriophage-fused epitopes to be used in the serodiagnosis of human VL. Eight phage clones were selected after the bio-panning rounds, and their reactivity was evaluated in a phage-ELISA assay against a human serological panel. A wild-type clone and the recombinant K39-based immunochromatographic test were used as controls. In the results, it was shown that all clones showed an excellent performance to serologically identify VL patients, demonstrating the feasibility of the isolated phages for developing a specific and sensitive serodiagnosis of human VL.

  16. Technological process and optimum design of organic materials vacuum pyrolysis and indium chlorinated separation from waste liquid crystal display panels.

    Science.gov (United States)

    Ma, En; Xu, Zhenming

    2013-12-15

    In this study, a technology process including vacuum pyrolysis and vacuum chlorinated separation was proposed to convert waste liquid crystal display (LCD) panels into useful resources using self-design apparatuses. The suitable pyrolysis temperature and pressure are determined as 300°C and 50 Pa at first. The organic parts of the panels were converted to oil (79.10 wt%) and gas (2.93 wt%). Then the technology of separating indium was optimized by central composite design (CCD) under response surface methodology (RSM). The results indicated the indium recovery ratio was 99.97% when the particle size is less than 0.16 mm, the weight percentage of NH4Cl to glass powder is 50 wt% and temperature is 450°C. The research results show that the organic materials, indium and glass of LCD panel can be recovered during the recovery process efficiently and eco-friendly.

  17. Information Phage Therapy Research Should Report

    OpenAIRE

    Stephen T. Abedon

    2017-01-01

    Bacteriophages, or phages, are viruses which infect bacteria. A large subset of phages infect bactericidally and, consequently, for nearly one hundred years have been employed as antibacterial agents both within and outside of medicine. Clinically these applications are described as phage or bacteriophage therapy. Alternatively, and especially in the treatment of environments, this practice instead may be described as a phage-mediated biocontrol of bacteria. Though the history of phage therap...

  18. Evaluation of helmet-mounted display targeting symbology based on eye tracking technology

    Science.gov (United States)

    Wang, Lijing; Wen, Fuzhen; Ma, Caixin; Zhao, Shengchu; Liu, Xiaodong

    2014-06-01

    The purpose of this paper is to find the Target Locator Lines (TLLs) which perform best by contrasting and comparing experiment based on three kinds of TTLs of fighter HMD. 10 university students, male, with an average age of 21-23, corrected visual acuity 1.5, participated in the experiment. In the experiment, head movement data was obtained by TrackIR. The geometric relationship between the coordinates of the real world and coordinates of the visual display was obtained by calculating the distance from viewpoint to midpoint of both eyes and the head movement data. Virtual helmet system simulation experiment environment was created by drawing TLLs of fighter HMD in the flight simulator visual scene. In the experiment, eye tracker was used to record the time and saccade trajectory. The results were evaluated by the duration of the time and saccade trajectory. The results showed that the symbol"locator line with digital vector length indication" cost most time and had the longest length of the saccade trajectory. It is the most ineffective and most unacceptable way. "Locator line with extending head vector length symbol" cost less time and had less length of the saccade trajectory. It is effective and acceptable;"Locator line with reflected vector length symbol" cost the least time and had the least length of the saccade trajectory. It is the most effective and most acceptable way. "Locator line with reflected vector length symbol" performs best. The results will provide reference value for the research of TTLs in future.

  19. Assessment of Anisotropic Semiconductor Nanorod and Nanoplatelet Heterostructures with Polarized Emission for Liquid Crystal Display Technology

    Energy Technology Data Exchange (ETDEWEB)

    Cunningham, Patrick D.; Souza, João B.; Fedin, Igor; She, Chunxing; Lee, Byeongdu; Talapin, Dmitri V.

    2016-06-28

    Semiconductor nanorods can emit linear-polarized light at efficiencies over 80%. Polarization of light in these systems, confirmed through single-rod spectroscopy, can be explained on the basis of the anisotropy of the transition dipole moment and dielectric confinement effects. Here we report emission polarization in macroscopic semiconductor polymer composite films containing CdSe/CdS nanorods and colloidal CdSe nanoplatelets. Anisotropic nanocrystals dispersed in polymer films of poly butyl-co-isobutyl methacrylate (PBiBMA) can be stretched mechanically in order to obtain unidirectionally aligned arrays. A high degree of alignment, corresponding to an orientation factor of 0.87, was achieved and large areas demonstrated polarized emission, with the contrast ratio I-parallel to/I-perpendicular to= 5.6, making these films viable candidates for use in liquid crystal display (LCD) devices. To some surprise, we observed significant optical anisotropy and emission polarization for 2D CdSe nanoplatelets with the electronic structure of quantum wells. The aligned nanorod arrays serve as optical funnels, absorbing unpolarized light and re-emitting light from deep-green to red with quantum efficiencies over 90% and high degree of linear polarization. Our results conclusively demonstrate the benefits of anisotropic nanostructures for LCD backlighting. The polymer films with aligned CdSe/CdS dot-in-rod and rod-in-rod nanostructures show more than 2-fold enhancement of brightness compared to the emitter layers with randomly oriented nanostructures. This effect can be explained as the combination of linearly polarized luminescence and directional emission from individual nanostructures.

  20. Environmental cues and genes involved in establishment of the superinfective Pf4 phage of Pseudomonas aeruginosa

    Directory of Open Access Journals (Sweden)

    Janice Gee Kay Hui

    2014-12-01

    Full Text Available Biofilm development in Pseudomonas aeruginosa is in part dependent on a filamentous phage, Pf4, which contributes to biofilm maturation, cell death, dispersal and variant formation, e.g. small colony variants. These biofilm phenotypes correlate with the conversion of the Pf4 phage into a superinfective variant that reinfects and kills the prophage carrying host, in contrast to other filamentous phage that normally replicate without killing their host. Here we have investigated the physiological cues and genes that may be responsible for this conversion. Flow through biofilms typically developed superinfective phage around day 4 or 5 of development and corresponded with dispersal. Starvation for carbon or nitrogen did not lead to the development of superinfective phage. In contrast, exposure of the biofilm to nitric oxide, H2O2 or the DNA damaging agent, mitomycin C, reproducibly led to an increase in the superinfective phage, suggesting that reactive oxygen or nitrogen species (RONS played a role in the formation of superinfective phage. In support of this, an oxyR mutant, the major oxidative stress regulator in P. aeruginosa, displayed significantly higher and earlier superinfection than the wild-type. Similarly, inactivation of mutS, a DNA mismatch repair gene, resulted in an early and a four log increase in the amount of superinfective phage generated by the biofilm. In contrast, loss of recA, important for DNA repair and SOS response, also resulted in a delayed and decreased production of superinfective phage. Treatments or mutations that increased superinfection also correlated with an increase in the production of morphotypic variants. The results suggest that the accumulation of RONS by the biofilm may result in DNA lesions in the Pf4 phage, leading to the formation of superinfective phage, which subsequently selects for morphotypic variants, such as small colony variants.

  1. In Vivo Selection of Phage Sequences and Characterization of Peptide-specific Binding to Breast Cancer Cells

    Institute of Scientific and Technical Information of China (English)

    Rui Wang; Ruifang Niu; Lin Zhang; Hongkai Zhang; Xiyin Wei; Yi Yang; Shiwu Zhang; Jing Wu; Min Wu; Youjia Cao

    2008-01-01

    OBJECTIVE To screen specific polypeptide target binding to breast cancer xenografts in vivo from a phage-displayed peptide library in order to provide peptide sequences for breast cancer tumor-targeting diagnosis and therapy.METHODS A mouse model for carrying breast cancer xenografts was established using Tientsin Albinao Ⅱ mice (TAII). A 12-peptide library was biopanned through 4 rounds.Phages were recovered and titrated from tumor xenografts and control tissue (liver). The distribution of phages was detected by immunohistochemical staining.RESULTS Phage homing to breast cancer was enriched through 4 rounds of biopanning, being 14-fold of that recovered from liver tissue. A peptide sequence, ASANPFPTKALL was characterized by randomly picked-up clones which appeared most frequently.Immunohistochemical staining revealed phage localization in cancer xenografts 40 min after injection of the enriched phages.When a specific phage was tested individually, the phage reclaimed from breast cancer xenografts was 14 times as those from control tissues.CONCLUSION Tumor-specific homing peptides may provide an effective tool for breast cancer target therapy. The in vivo phage display selection technique employed in this study was feasible and applicable to screening peptides that home to.breast cells.

  2. Human Volunteers Receiving Escherichia coli Phage T4 Orally: a Safety Test of Phage Therapy

    OpenAIRE

    Bruttin, Anne; Brüssow, Harald

    2005-01-01

    Fifteen healthy adult volunteers received in their drinking water a lower Escherichia coli phage T4 dose (103 PFU/ml), a higher phage dose (105 PFU/ml), and placebo. Fecal coliphage was detected in a dose-dependent way in volunteers orally exposed to phage. All volunteers receiving the higher phage dose showed fecal phage 1 day after exposure; this prevalence was only 50% in subjects receiving the lower phage dose. No fecal phage was detectable a week after a 2-day course of oral phage applic...

  3. A novel screening system for claudin binder using baculoviral display.

    Directory of Open Access Journals (Sweden)

    Hideki Kakutani

    Full Text Available Recent progress in cell biology has provided new insight into the claudin (CL family of integral membrane proteins, which contains more than 20 members, as a target for pharmaceutical therapy. Few ligands for CL have been identified because it is difficult to prepare CL in an intact form. In the present study, we developed a method to screen for CL binders by using the budded baculovirus (BV display system. CL4-displaying BV interacted with a CL4 binder, the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE, but it did not interact with C-CPE that was mutated in its CL4-binding region. C-CPE did not interact with BV and CL1-displaying BV. We used CL4-displaying BV to select CL4-binding phage in a mixture of a scFv-phage and C-CPE-phage. The percentage of C-CPE-phage in the phage mixture increased from 16.7% before selection to 92% after selection, indicating that CL-displaying BV may be useful for the selection of CL binders. We prepared a C-CPE phage library by mutating the functional amino acids. We screened the library for CL4 binders by affinity to CL4-displaying BV, and we found that the novel CL4 binders modulated the tight-junction barrier. These findings indicate that the CL-displaying BV system may be a promising method to produce a novel CL binder and modulator.

  4. [Novel bioconversion systems using a yeast molecular display system].

    Science.gov (United States)

    Shibasaki, Seiji

    2010-11-01

    The budding yeast Saccharomyces cerevisiae has been used for the process of fermentation as well as for studies in biochemistry and molecular biology as a eukaryotic model cell or tool for the analysis of gene functions. Thus, yeast is essential in industries and researches. Yeast cells have a cell wall, which is one characteristic that helps distinguish yeast cells from other eukaryotic cells such as mammalian cells. We have developed a molecular display system using the protein of the yeast cell wall as an anchor for foreign proteins. Yeast cells have been designed for use in sensing and metal adsorption, and have been used in vaccines and for screening novel proteins. Currently, yeast is used not only as a tool for analyzing gene or protein function but also in molecular display technology. The phage display system, which is at the forefront of molecular display technologies, is a powerful tool for screening ligands bound to a target molecule and for analyzing protein-protein interactions; however, in some cases, eukaryotic proteins are not easily expressed by this system. On the other hand, yeast cells have the ability to express eukaryotic proteins and proliferate; thus, these cells display various proteins. Yeast cells are more appropriate for white biotechnology. In this review, displays of enzymes that are important in bioconversion, such as lipases and β-glucosidases, are going to be introduced.

  5. Biochemical analysis and application of molecular display technology on Candida albicans for diagnosing and preventing candidiasis.

    Science.gov (United States)

    Shibasaki, Seiji; Aoki, Wataru; Ueda, Mitsuyoshi

    2013-01-01

    Medical facilities and advances in therapeutics have improved world over in recent times. Concomitant with this, the human population has been growing steadily. However, emerging infectious diseases such as severe acute respiratory syndrome (SARS) and AIDS, as well as re-emerging infectious diseases such as Japanese encephalitis and dengue fever, have been spreading in recent times. Three major infectious diseases, namely AIDS, malaria, and tuberculosis, are killing around 8 million people in the world annually. Although drugs effective against these infectious diseases are available at present, drastic therapeutics have not been developed yet. In addition, vaccines against these diseases often cannot prevent infections, because pathogenic viruses or bacteria evade the immune system of the host. Many diseases and emerging infections of pathogenic bacteria cannot be controlled by conventional pharmaceutics. These pathogens secrete regulatory factors. When the produced regulatory factor attains a certain level, an active factor is then produced by the pathogen to destroy the host. Considering these phenomena, we thought investigating characteristic regulatory or active factors will pave the way for developing novel vaccines or diagnostic drugs. Therefore, candidiasis was selected as a model, and application of the secretory protease of Candida albicans was examined for the development of novel drugs. Screening of novel candidates of antigens of C. albicans and vaccine development are also underway. In this paper, our strategy of platform technology against various infectious diseases are introduced.

  6. Phage nanofibers induce vascularized osteogenesis in 3D printed bone scaffolds.

    Science.gov (United States)

    Wang, Jianglin; Yang, Mingying; Zhu, Ye; Wang, Lin; Tomsia, Antoni P; Mao, Chuanbin

    2014-08-01

    A virus-activated matrix is developed to overcome the challenge of forming vascularized bone tissue. It is generated by filling a 3D printed bioceramic scaffold with phage nanofibers displaying high-density RGD peptide. After it is seeded with mesenchymal stem cells (MSCs) and implanted into a bone defect, the phage nanofibers induce osteogenesis and angiogenesis by activating endothelialization and osteogenic differentiation of MSCs.

  7. Clinical aspects of phage therapy.

    Science.gov (United States)

    Międzybrodzki, Ryszard; Borysowski, Jan; Weber-Dąbrowska, Beata; Fortuna, Wojciech; Letkiewicz, Sławomir; Szufnarowski, Krzysztof; Pawełczyk, Zdzisław; Rogóż, Paweł; Kłak, Marlena; Wojtasik, Elżbieta; Górski, Andrzej

    2012-01-01

    Phage therapy (PT) is a unique method of treatment of bacterial infections using bacteriophages (phages)-viruses that specifically kill bacteria, including their antibiotic-resistant strains. Over the last decade a marked increase in interest in the therapeutic use of phages has been observed, which has resulted from a substantial rise in the prevalence of antibiotic resistance of bacteria, coupled with an inadequate number of new antibiotics. The first, and so far the only, center of PT in the European Union is the Phage Therapy Unit (PTU) established at the Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Wrocław, Poland in 2005. This center continues the rich tradition of PT in Poland, which dates from the early 1920s. The main objective of this chapter is to present a detailed retrospective analysis of the results of PT of 153 patients with a wide range of infections resistant to antibiotic therapy admitted for treatment at the PTU between January 2008 and December 2010. Analysis includes the evaluation of both the efficacy and the safety of PT. In general, data suggest that PT can provide good clinical results in a significant cohort of patients with otherwise untreatable chronic bacterial infections and is essentially well tolerated. In addition, the whole complex procedure employed to obtain and characterize therapeutic phage preparations, as well as ethical aspects of PT, is discussed.

  8. A zero-footprint 3D visualization system utilizing mobile display technology for timely evaluation of stroke patients

    Science.gov (United States)

    Park, Young Woo; Guo, Bing; Mogensen, Monique; Wang, Kevin; Law, Meng; Liu, Brent

    2010-03-01

    When a patient is accepted in the emergency room suspected of stroke, time is of the utmost importance. The infarct brain area suffers irreparable damage as soon as three hours after the onset of stroke symptoms. A CT scan is one of standard first line of investigations with imaging and is crucial to identify and properly triage stroke cases. The availability of an expert Radiologist in the emergency environment to diagnose the stroke patient in a timely manner only increases the challenges within the clinical workflow. Therefore, a truly zero-footprint web-based system with powerful advanced visualization tools for volumetric imaging including 2D. MIP/MPR, 3D display can greatly facilitate this dynamic clinical workflow for stroke patients. Together with mobile technology, the proper visualization tools can be delivered at the point of decision anywhere and anytime. We will present a small pilot project to evaluate the use of mobile technologies using devices such as iPhones in evaluating stroke patients. The results of the evaluation as well as any challenges in setting up the system will also be discussed.

  9. Complete Genome Sequence of Vibrio anguillarum Phage CHOED Successfully Used for Phage Therapy in Aquaculture

    OpenAIRE

    Romero, Jaime; Higuera, Gastón; Gajardo,Felipe; Castillo, Daniel; Middleboe, Mathias; García, Katherine; Ramírez, Carolina; Espejo, Romilio T.

    2014-01-01

    Vibrio anguillarum phage CHOED was isolated from Chilean mussels. It is a virulent phage showing effective inhibition of V. anguillarum. CHOED has potential in phage therapy, because it can protect fish from vibriosis in fish farms. Here, we announce the completely sequenced genome of V. anguillarum phage CHOED.

  10. Information Phage Therapy Research Should Report

    National Research Council Canada - National Science Library

    Stephen T Abedon

    2017-01-01

    .... Clinically these applications are described as phage or bacteriophage therapy. Alternatively, and especially in the treatment of environments, this practice instead may be described as a phage-mediated biocontrol of bacteria...

  11. Phage Therapy: Eco-Physiological Pharmacology

    OpenAIRE

    Abedon, Stephen T.

    2014-01-01

    Bacterial virus use as antibacterial agents, in the guise of what is commonly known as phage therapy, is an inherently physiological, ecological, and also pharmacological process. Physiologically we can consider metabolic properties of phage infections of bacteria and variation in those properties as a function of preexisting bacterial states. In addition, there are patient responses to pathogenesis, patient responses to phage infections of pathogens, and also patient responses to phage virio...

  12. Naked Eye 3D Technology of Full Color LED Display Screen%浅谈全彩LED显示屏的裸眼3D技术

    Institute of Scientific and Technical Information of China (English)

    马小华

    2014-01-01

    This paper introduces the 3D stereo vision principle and 3 Naked-eye 3D display technology is applied to the principle of LED display screen and application method, several 3D autostereoscopic display quality technology application in the display of the LED potential analysis and research and discussion.%本文介绍了3D立体视觉原理以及三种裸眼3D显示技术应用到LED显示屏上的原理以及应用方法,对几种裸眼3D立体显示技术应用在LED显示屏上的优劣势进行分析和研究探讨。

  13. Identification of Posttranslational Modification-Dependent Protein Interactions Using Yeast Surface Displayed Human Proteome Libraries.

    Science.gov (United States)

    Bidlingmaier, Scott; Liu, Bin

    2015-01-01

    The identification of proteins that interact specifically with posttranslational modifications such as phosphorylation is often necessary to understand cellular signaling pathways. Numerous methods for identifying proteins that interact with posttranslational modifications have been utilized, including affinity-based purification and analysis, protein microarrays, phage display, and tethered catalysis. Although these techniques have been used successfully, each has limitations. Recently, yeast surface-displayed human proteome libraries have been utilized to identify protein fragments with affinity for various target molecules, including phosphorylated peptides. When coupled with fluorescently activated cell sorting and high throughput methods for the analysis of selection outputs, yeast surface-displayed human proteome libraries can rapidly and efficiently identify protein fragments with affinity for any soluble ligand that can be fluorescently detected, including posttranslational modifications. In this review we compare the use of yeast surface display libraries to other methods for the identification of interactions between proteins and posttranslational modifications and discuss future applications of the technology.

  14. Phage therapy--constraints and possibilities.

    Science.gov (United States)

    Nilsson, Anders S

    2014-05-01

    The rise of antibiotic-resistant bacterial strains, causing intractable infections, has resulted in an increased interest in phage therapy. Phage therapy preceded antibiotic treatment against bacterial infections and involves the use of bacteriophages, bacterial viruses, to fight bacteria. Virulent phages are abundant and have proven to be very effective in vitro, where they in most cases lyse any bacteria within the hour. Clinical trials on animals and humans show promising results but also that the treatments are not completely effective. This is partly due to the studies being carried out with few phages, and with limited experimental groups, but also the fact that phage therapy has limitations in vivo. Phages are large compared with small antibiotic molecules, and each phage can only infect one or a few bacterial strains. A very large number of different phages are needed to treat infections as these are caused by genetically different strains of bacteria. Phages are effective only if enough of them can reach the bacteria and increase in number in situ. Taken together, this entails high demands on resources for the construction of phage libraries and the testing of individual phages. The effectiveness and host range must be characterized, and immunological risks must be assessed for every single phage.

  15. Screening of large molecule diversities by phage display

    National Research Council Canada - National Science Library

    Rentero, Inmaculada; Heinis, Christian

    2011-01-01

    Molecules with tailored binding specificities are needed for many purposes such as the development of therapeutics, the detection and purification of biomolecules or in chemical biology for the study...

  16. Pitfalls to avoid when using phage display for snake toxins

    DEFF Research Database (Denmark)

    Laustsen, Andreas Hougaard; Lauridsen, Line Præst; Lomonte, Bruno

    2017-01-01

    Antivenoms against bites and stings from snakes, spiders, and scorpions are associated with immunological side effects and high cost of production, since these therapies are still derived from the serum of hyper-immunized production animals. Biotechnological innovations within envenoming therapies...

  17. Phage display-derived human antibodies against specific glycosaminoglycan epitopes.

    NARCIS (Netherlands)

    Smits, N.C.; Lensen, J.F.M.; Wijnhoven, T.J.M.; Dam, G.B. ten; Jenniskens, G.J.; Kuppevelt, A.H.M.S.M. van

    2006-01-01

    Glycosaminoglycans (GAGs) are long unbranched polysaccharides, most of which are linked to a core protein to form proteoglycans. Depending on the nature of their backbone, one can discern galactosaminoglycans (chondroitin sulfate [CS] and dermatan sulfate [DS]) and glucosaminoglycans (heparan sulfat

  18. A blended learning concept for an engineering course in the field of color representation and display technologies

    Science.gov (United States)

    Vauderwange, Oliver; Wozniak, Peter; Javahiraly, Nicolas; Curticapean, Dan

    2016-09-01

    The Paper presents the design and development of a blended learning concept for an engineering course in the field of color representation and display technologies. A suitable learning environment is crucial for the success of the teaching scenario. A mixture of theoretical lectures and hands-on activities with practical applications and experiments, combined with the advantages of modern digital media is the main topic of the paper. Blended learning describes the didactical change of attendance periods and online periods. The e-learning environment for the online period is designed toward an easy access and interaction. Present digital media extends the established teaching scenarios and enables the presentation of videos, animations and augmented reality (AR). Visualizations are effective tools to impart learning contents with lasting effect. The preparation and evaluation of the theoretical lectures and the hands-on activities are stimulated and affects positively the attendance periods. The tasks and experiments require the students to work independently and to develop individual solution strategies. This engages and motivates the students, deepens the knowledge. The authors will present their experience with the implemented blended learning scenario in this field of optics and photonics. All aspects of the learning environment will be introduced.

  19. Phage typing of Staphylococcus saprophyticus.

    Science.gov (United States)

    Torres Pereira, A.; Melo Cristino, J. A.

    1991-01-01

    This study included 502 staphylococcus strains; Staphylococcus saprophyticus (297 strains) S. cohnii (47), S. xylosus (10), S. epidermidis (67) and S. aureus (81). Mitomycin C induction was performed on 100 isolates of S. saprophyticus and all induced strains were reacted with each other. Twenty-six strains proved to be lysogenic. Phages were propagated and titrated. With 12 of the phages there were three frequent associations, named lytic groups A, B and C, which included 75% of all typable strains. Typability of the system was 45% and reproducibility was between 94.2% and 100%. Phages did not lyse S. aureus and S. epidermidis strains, but they lysed S. saprophyticus and only rare strains of other novobiocin resistant species. Effective S. saprophyticus typing serves ecological purposes and tracing the origin of urinary strains from the skin or mucous membranes. Phage typing in association with plasmid profiling previously described, are anticipated as complementary methods with strong discriminatory power for differentiating among S. saprophyticus strains. PMID:1752305

  20. Phage lytic enzymes: a history

    Institute of Scientific and Technical Information of China (English)

    David; Trudil

    2015-01-01

    There are many recent studies regarding the efficacy of bacteriophage-related lytic enzymes: the enzymes of ‘bacteria-eaters’ or viruses that infect bacteria. By degrading the cell wall of the targeted bacteria, these lytic enzymes have been shown to efficiently lyse Gram-positive bacteria without affecting normal flora and non-related bacteria. Recent studies have suggested approaches for lysing Gram-negative bacteria as well(Briersa Y, et al., 2014). These enzymes include: phage-lysozyme, endolysin, lysozyme, lysin, phage lysin, phage lytic enzymes, phageassociated enzymes, enzybiotics, muralysin, muramidase, virolysin and designations such as Ply, PAE and others. Bacteriophages are viruses that kill bacteria, do not contribute to antimicrobial resistance, are easy to develop, inexpensive to manufacture and safe for humans, animals and the environment. The current focus on lytic enzymes has been on their use as anti-infectives in humans and more recently in agricultural research models. The initial translational application of lytic enzymes, however, was not associated with treating or preventing a specifi c disease but rather as an extraction method to be incorporated in a rapid bacterial detection assay(Bernstein D, 1997).The current review traces the translational history of phage lytic enzymes–from their initial discovery in 1986 for the rapid detection of group A streptococcus in clinical specimens to evolving applications in the detection and prevention of disease in humans and in agriculture.

  1. Efficient method to optimize antibodies using avian leukosis virus display and eukaryotic cells.

    Science.gov (United States)

    Yu, Changming; Pike, Gennett M; Rinkoski, Tommy A; Correia, Cristina; Kaufmann, Scott H; Federspiel, Mark J

    2015-08-11

    Antibody-based therapeutics have now had success in the clinic. The affinity and specificity of the antibody for the target ligand determines the specificity of therapeutic delivery and off-target side effects. The discovery and optimization of high-affinity antibodies to important therapeutic targets could be significantly improved by the availability of a robust, eukaryotic display technology comparable to phage display that would overcome the protein translation limitations of microorganisms. The use of eukaryotic cells would improve the diversity of the displayed antibodies that can be screened and optimized as well as more seamlessly transition into a large-scale mammalian expression system for clinical production. In this study, we demonstrate that the replication and polypeptide display characteristics of a eukaryotic retrovirus, avian leukosis virus (ALV), offers a robust, eukaryotic version of bacteriophage display. The binding affinity of a model single-chain Fv antibody was optimized by using ALV display, improving affinity >2,000-fold, from micromolar to picomolar levels. We believe ALV display provides an extension to antibody display on microorganisms and offers virus and cell display platforms in a eukaryotic expression system. ALV display should enable an improvement in the diversity of properly processed and functional antibody variants that can be screened and affinity-optimized to improve promising antibody candidates.

  2. Transfer of antibiotic-resistance genes via phage-related mobile elements.

    Science.gov (United States)

    Brown-Jaque, Maryury; Calero-Cáceres, William; Muniesa, Maite

    2015-05-01

    Antibiotic resistance is a major concern for society because it threatens the effective prevention of infectious diseases. While some bacterial strains display intrinsic resistance, others achieve antibiotic resistance by mutation, by the recombination of foreign DNA into the chromosome or by horizontal gene acquisition. In many cases, these three mechanisms operate together. Several mobile genetic elements (MGEs) have been reported to mobilize different types of resistance genes and despite sharing common features, they are often considered and studied separately. Bacteriophages and phage-related particles have recently been highlighted as MGEs that transfer antibiotic resistance. This review focuses on phages, phage-related elements and on composite MGEs (phages-MGEs) involved in antibiotic resistance mobility. We review common features of these elements, rather than differences, and provide a broad overview of the antibiotic resistance transfer mechanisms observed in nature, which is a necessary first step to controlling them.