Academic literature on the topic 'Phage displayed scFv'
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Journal articles on the topic "Phage displayed scFv"
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Zhao, Peng, Guijie Zhu, Lihua Zhang, Zhen Liang, Zonghai Li, and Yukui Zhang. "New method for determination of average scFv fragment number displayed on the M13 phage surface." Pure and Applied Chemistry 82, no. 1 (January 3, 2010): 205–11. http://dx.doi.org/10.1351/pac-con-09-02-03.
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Single-chain-Fv (scFv) display M13 phage library has been regarded as a powerful tool for screening specific antibodies via binding with target proteins. Generally, the library quality is evaluated through detecting gene fragments by molecular biology methods, which is not only time- and labor-consuming, but also impossible to obtain quantitative information about the binding capacity of the phage library. In our recent study, a new method to calculate the average scFv number displayed on the M13 phage surface was proposed by capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection. By this method, enhanced green fluorescent protein (EGFP) and scFv phage clones that could specifically bind with EGFP were mixed with different ratios, followed by analysis by CE-LIF. With the dilution of EGFP by phage solution, the peak areas of scFv phage clones and free EGFP were decreased continuously, while that of the EGFP-M13 phage complex was found to decrease initially, then trend to be stable, and finally decrease further. When the volume ratio of the M13 phage to EGFP reached 660:1, corresponding to the molecule number ratio as 1:2.6, no more EGFP was found to bind with the M13 phage, which demonstrated that, by average, 2.6 scFv fragments that could bind with EGFP were displayed on the M13 phage surface. All these experimental results demonstrated that, by such a method, the quantitative evaluation of the phage library could be achieved with high throughput and accuracy.
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Brettschneider, Kerstin, Anja Naumann, Sonja Neimanis, Joerg Kahle, Christine Heller, Thomas Klingebiel, Dirk Schwabe, and Christoph Koenigs. "Functional Analysis of Phage Display Selected Single-Chain Variable Antibody Fragments (scFvs) Specific for Anti-FVIII Antibodies." Blood 124, no. 21 (December 6, 2014): 1499. http://dx.doi.org/10.1182/blood.v124.21.1499.1499.
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Abstract The development of inhibitory antibodies against coagulation factor VIII (FVIII) is currently the most serious complication for hemophilia A patients that undergo FVIII replacement therapy. In addition, non-hemophilia A patients can spontaneously develop inhibitory auto-antibodies to FVIII, which results in acquired haemophilia A. The control of the allo- or autoimmune response to FVIII apparently includes the elicitation of anti-idiotypic antibodies. In this study the capacity of anti-idiotypic single-chain variable antibody fragments (scFvs) for neutralization of inhibitory anti-FVIII antibodies (FVIII inhibitors) was evaluated in vitro and in vivo. Anti-idiotypic scFvs were selected from phage-displayed libraries against murine monoclonal FVIII-specific inhibitors. As the majority of inhibitory antibodies is directed against the A2 or C2 domain of FVIII, strongly inhibitory A2- and C2-specific antibodies served as targets. Selected scFvs were expressed as scFv-Fc fusion proteins. Analysis of the scFv-Fcs by ELISA confirmed the specific binding to the cognate targets and binding studies via surface plasmon resonance revealed high affinities within the nanomolar range. Further characterization showed that binding of inhibitors to immobilized FVIII was blocked by specific scFv-Fcs in vitro. The ability of scFv-Fcs to neutralize their corresponding inhibitors was analyzed in a functional clotting assay. By adding scFv-Fcs to plasma spiked with inhibitors, FVIII activity was restored to 80% in a concentration dependent manner. FVIII knockout mice served as model organism for testing the capacity of scFv-Fcs to restore coagulation in vivo. Subsequent injection of FVIII following the injection of the inhibitors resulted in a largely reduced FVIII activity. However, FVIII activity was recovered in a concentration dependent manner by adding cognate anti-idiotypes. The scFv-Fcs were either preincubated with the corresponding inhibitor or added to the FVIII mixture without preincubation. The latter represents an adaption to a therapeutic setting. In conclusion, phage display selected anti-idiotypic scFvs are able to bind and effectively neutralize their target inhibitors in vitro and in vivo. Based on these promising results the potential of anti-idiotypic scFvs for the development of specific cell based immunotherapies for hemophilia A patients with inhibitors is currently under investigation. Disclosures No relevant conflicts of interest to declare.
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Goswami, Pooja, Deepti Saini, and Subrata Sinha. "Phage Displayed scFv: pIII Scaffold May Fine Tune Binding Specificity." Hybridoma 28, no. 5 (October 2009): 327–31. http://dx.doi.org/10.1089/hyb.2009.0008.
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Muller, Bruno H., Florence Lafay, Caroline Demangel, Pierre Perrin, Noël Tordo, Anne Flamand, Pierre Lafaye, and Jean-Luc Guesdon. "Phage-displayed and soluble mouse scFv fragments neutralize rabies virus." Journal of Virological Methods 67, no. 2 (September 1997): 221–33. http://dx.doi.org/10.1016/s0166-0934(97)00099-2.
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Wind, Troels, Brian Stausbøl-Grøn, Svend Kjær, Liselotte Kahns, Kristian H. Jensen, and Brian F. C. Clark. "Retrieval of phage displayed scFv fragments using direct bacterial elution." Journal of Immunological Methods 209, no. 1 (November 1997): 75–83. http://dx.doi.org/10.1016/s0022-1759(97)00151-8.
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van den Brink, Edward N., Ellen A. M. Turenhout, Julian Davies, Niels Bovenschen, Karin Fijnvandraat, Willem H. Ouwehand, Marjolein Peters, and Jan Voorberg. "Human antibodies with specificity for the C2 domain of factor VIII are derived from VH1 germline genes." Blood 95, no. 2 (January 15, 2000): 558–63. http://dx.doi.org/10.1182/blood.v95.2.558.
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A serious complication in hemophilia care is the development of factor VIII (FVIII) neutralizing antibodies (inhibitors). The authors used V gene phage display technology to define human anti-FVIII antibodies at the molecular level. The IgG4-specific, variable, heavy-chain gene repertoire of a patient with acquired hemophilia was combined with a nonimmune, variable, light-chain gene repertoire for display as single-chain variable domain antibody fragments (scFv) on filamentous phage. ScFv were selected by 4 rounds of panning on immobilized FVIII light chain. Sequence analysis revealed that isolated scFv were characterized by VH domains encoded by germline genes DP-10, DP-14, and DP-88, all belonging to the VH1 gene family. All clones displayed extensive hypermutation and were characterized by unusually long CDR3 sequences of 20 to 23 amino acids. Immunoprecipitation revealed that all scFv examined bound to the C2 domain of FVIII. Furthermore, isolated scFv competed with an inhibitory murine monoclonal antibody for binding to the C2 domain. Even though scFv bound FVIII with high affinity, they did not inhibit FVIII activity. Interestingly, the addition of scFv diminished the inhibitory potential of patient-derived antibodies with C2 domain specificity. These results suggest that the epitope of a significant portion of anti-C2 domain antibodies overlaps with that of the scFv isolated in this study.
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Naumann, Anja, Jörg Kahle, Nadia Reiss, Dirk Schwabe, Christine Heller, Thomas Klingebiel, and Christoph Königs. "Development of a Factor VIII-Specific Immunotherapy for Hemophilia A." Blood 120, no. 21 (November 16, 2012): 3361. http://dx.doi.org/10.1182/blood.v120.21.3361.3361.
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Abstract Abstract 3361 The development of neutralizing allo-antibodies against coagulation factor VIII (FVIII) is currently the most serious complication for hemophilia A patients that undergo FVIII replacement therapy. Non-hemophiliacs can spontaneously develop inhibitory auto-antibodies to FVIII, which results in a condition called acquired hemophilia A. The control of the allo- or autoimmune response to FVIII apparently includes the elicitation of an anti-idiotypic immune response. Immune tolerance induction (ITI) by frequent administration of high doses of FVIII is successful in most patients. The remaining up to 20% failing ITI face increased morbidity and mortality. To elucidate the capacity of single-chain variable antibody fragments (scFvs) for neutralization of inhibitory anti-FVIII antibodies (FVIII inhibitors), scFvs that specifically interact with strong inhibitory murine monoclonal anti-human FVIII antibodies (anti-hFVIII mAbs) were selected by screening synthetic scFv phage displayed libraries. Since the majority of inhibitory anti-hFVIII mAbs are directed against the FVIII A2 or C2 domain, selection of scFvs was performed for two anti-A2 and two anti-C2 mAbs. Affinity selection identified several specific, potential anti-idiotypic scFvs for each anti-hFVIII mAb, which were further analyzed. ScFvs expressed as IgG fusion protein bind to mAbs with affinities up to 0,1nM. Binding of mAbs to immobilized FVIII was inhibited via specific scFvs by more than 90%. In the functional Bethesda Assay FVIII activity was fully restored when corresponding anti-idiotypic scFvs were added to plasma spiked with FVIII-specific mAbs. In addition, the cross-reactivity of scFvs with heterologous mAbs specific for A2 and C2 was tested and confirmed the exclusive interaction of the selected scFvs with their respective mAb. Overall, anti-idiotypic scFvs specific for anti-hFVIII mAbs can be successfully selected from phage displayed libraries and efficiently neutralize FVIII inhibitors. ScFv anti-idiotypes may therefore facilitate the development of specific immunotherapies for hemophilia patients with inhibitors. Disclosures: No relevant conflicts of interest to declare.
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Stuknytė, Milda, Eeva-Christine Brockmann, Tuomas Huovinen, Simone Guglielmetti, Diego Mora, Valentina Taverniti, Stefania Arioli, Ivano De Noni, and Urpo Lamminmäki. "Lactobacillus helveticus MIMLh5-Specific Antibodies for Detection of S-Layer Protein in Grana Padano Protected-Designation-of-Origin Cheese." Applied and Environmental Microbiology 80, no. 2 (November 15, 2013): 694–703. http://dx.doi.org/10.1128/aem.03057-13.
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ABSTRACTSingle-chain variable-fragment antibodies (scFvs) have considerable potential in immunological detection and localization of bacterial surface structures. In this study, synthetic phage-displayed antibody libraries were used to select scFvs against immunologically active S-layer protein ofLactobacillus helveticusMIMLh5. After three rounds of panning, five relevant phage clones were obtained, of which four were specific for the S-layer protein ofL. helveticusMIMLh5 and one was also capable of binding to the S-layer protein ofL. helveticusATCC 15009. All five anti-S-layer scFvs were expressed inEscherichia coliXL1-Blue, and their specificity profiles were characterized by Western blotting. The anti-S-layer scFv PolyH4, with the highest specificity for the S-layer protein ofL. helveticusMIMLh5, was used to detect the S-layer protein in Grana Padano protected-designation-of-origin (PDO) cheese extracts by Western blotting. These results showed promising applications of this monoclonal antibody for the detection of immunomodulatory S-layer protein in dairy (and dairy-based) foods.
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Schmidt, Anja, Kerstin Brettschneider, Jörg Kahle, Aleksander Orlowski, Karin Becker-Peters, Diana Stichel, Jörg Schulze, et al. "Neutralisation of factor VIII inhibitors by anti-idiotypes isolated from phage-displayed libraries." Thrombosis and Haemostasis 116, no. 07 (January 2016): 32–41. http://dx.doi.org/10.1160/th15-12-0925.
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SummaryFollowing replacement therapy with coagulation factor VIII (FVIII), up to 30 % of haemophilia A patients develop FVIII-specific inhibitory antibodies (FVIII inhibitors). Immune tolerance induction (ITI) is not always successful, resulting in a need for alternative treatments for FVIII inhibitor-positive patients. As tolerance induction in the course of ITI appears to involve the formation of anti-idiotypes specific for anti-FVIII antibodies, such anti-idiotypes might be used to restore haemostasis in haemophilia A patients with FVIII inhibitors. We isolated antiidiotypic antibody fragments (scFvs) binding to murine FVIII inhibitors 2-76 and 2-77 from phage-displayed libraries. FVIII inhibitor/anti-idiotype interactions were very specific as no cross-reactivity with other FVIII inhibitors or isotype controls was observed. ScFvs blocked binding of FVIII inhibitors to FVIII and neutralised their cognate inhibitors in vitro and a monoclonal mouse model. In addition, scFv JkH5 specific for FVIII inhibitor 2-76 stained 2-76-producing hybridoma cells. JkH5 residues R52 and Y226, located in complementary determining regions, were identified as crucial for the JkH5/2-76 interaction using JkH5 alanine mutants. SPR spectroscopy revealed that JkH5 interacts with FVIII inhibitor 2-76 with nanomolar affinity. Thus, FVIII inhibitorspecific, high-affinity anti-idiotypes can be isolated from phagedisplayed libraries and neutralise their respective inhibitors. Furthermore, we show that anti-idiotypic scFvs might be utilised to specifically target inhibitor-specific B cells. Hence, a pool of anti-idiotypes could enable the reestablishment of haemostasis in the presence of FVIII inhibitors in patients or even allow the depletion of inhibitors by targeting inhibitor-specific B cell populations.
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Massion, P. P., T. V. Pedchenko, D. V. Parekh, and R. Mernaugh. "Selection of lung cancer-associated scFv antibodies from cultured cells." Journal of Clinical Oncology 27, no. 15_suppl (May 20, 2009): e22137-e22137. http://dx.doi.org/10.1200/jco.2009.27.15_suppl.e22137.
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e22137 Background: Lung cancer is the most common cause of cancer-related deaths in the world. There is a critical need for new strategies of early lung cancer detection. The identification of tumor-associated antigens and corresponding antibodies is one approach to discovery of diagnostic biomarkers. We used a large phage-displayed recombinant antibody library and normal human lung epithelial and non-small cell lung cancer cell lines to select for and identify recombinant antibodies specific for proteins expressed, or over-expressed, in lung cancer. Methods: The antibody library was used to select for recombinant scFv antibodies reactive with proteins present, or aberrantly expressed, in non-small cell lung cancer cell lines (A549, H549, H157, H23) in comparison to normal lung cell lines (BEAS-2B, 16-HBE, KT). Soluble scFv antibodies were obtained through 2 rounds of phage antibody cross-absorption (on normal cell lines) and selection (on non-small lung cancer cell lines). Soluble scFv were assayed by a high-throughput fluorometric microvolume assay technology (FMAT) against normal and cancer lung cell line proteins. ScFv antibodies selected by FMAT were evaluated further with Western blot-based assays. Results: More than 100 scFv antibodies identified by FMAT bound preferentially to proteins in lung cancer. Of these, 46 scFv were assayed by a high throughput Western slot blot immunoassay against pooled normal lung and lung cancer cell lysates. Eight scFv were assayed in Western blot against individual lung normal and non-small lung cancer cell line lysates. Four of these demonstrated differential binding to normal and cancer cell lysates. Conclusions: In summary, we were able to detect cancer-associated antigens in lung cancer cell lines using a phage display antibody library. In combination with high-throughput fluorescent and Western blot assays, four unique scFv antibodies were selected that differentially bound to normal and lung cancer cell lysates. These scFv will be tested as candidate biomarkers of lung cancer in independent tissue and serum samples from patient with and without lung cancer to determine utility for use in lung cancer diagnosis. No significant financial relationships to disclose.
Dissertations / Theses on the topic "Phage displayed scFv"
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Gomes, Carlos Henrique Rodrigues. "Construção de uma bibilioteca de anticorpos ScFv dirigidos contra o fator de crescimento vascular (VEGF)." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-09082013-093807/.
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Angiogênese é a formação de novos vasos sanguíneos a partir de vasos já existentes e é importante em processos fisiológicos, que em adultos é restrita ao reparo tecidual e ao ciclo reprodutivo feminino. Entretanto, doenças, como câncer ou retinopatias, induzem a formação da angiogênese patológica, necessária para a progressão destas patologias. Anticorpos monoclonais constituem uma das classes de biofármacos que mais cresce e com impacto importante nas doenças dependentes de angiogênese. Entre as diversas metodologias para a identificação de anticorpos monoclonais contra alvos terapêuticos, está o phage display. Por causa do fator de crescimento endotelial vascular (VEGF) ser o principal fator responsável pela formação de novos vasos, o principal biofármaco anti-angiogênico disponível na clínica atualmente é um anticorpo monoclonal (bevacizumab) direcionadas contra o VEGF. Embora terapias anti-VEGF sejam eficazes, ainda não são ideais devido a efeitos colaterais indesejados e a resistência medicamentosa. Novas alternativas são necessárias a fim de aperfeiçoar as terapias angiogênicas. O objetivo do nosso estudo é identificar novos alvos moleculares e desenvolver novos agentes terapêuticos para doenças dependentes de angiogênese. Para atingirmos nossa meta escolhemos o sistema de phage display para selecionarmos anticorpos com propriedades angiogênicas. Uma biblioteca de de anticorpos foi desenvolvida em nosso laboratório, dirigida contra a molécula VEGF, em particular uma de suas isoformas. Os animais imunizados desenvolveram anticorpos específicos, detectados por ELISA e Western-blot. A amplificação do pool de genes das cadeias leve e pesada de imunoglobulinas foi realizada para produzir os fragmentos single-chain (ScFv) que foram então clonados no vetor para a construção da biblioteca. A biblioteca de display de anticorpos ScFv será, portanto, analisada em plataformas angiogênicas para isolar anticorpos específicos contra isoformas de VEGF e novos marcadores moleculares de superfície celular expressos por células endoteliais ativadas.Angiogenesis is the formation of new blood vessels from pre-existing ones and is an important physiological process, which in adults is mostly restricted to wound healing or the female reproductive cycle. However, different illnesses, such as cancer or retinopathies, induce the formation of pathological angiogenesis, necessary for disease progression. Monoclonal antibodies are one of the fastest growing class of biopharmaceuticals with important implications in angiogenesis dependent diseases. Among various methods for the identification of monoclonal antibodies against therapeutic targets is phage display technology. Because the vascular endothelial growth factor (VEGF) is the main molecular factor responsible for the formation of new blood vessels, the major anti-angiogenic drug available in the clinic today is a monoclonal antibody (bevacizumab) directed against VEGF. However, although anti-VEGF therapies are effective, they are not yet ideal due to undesirable side effects and drug resistance. Novel alternatives are necessary to improve on angiogenic therapies. The aim of our study is to identify novel molecular targets and to develop new therapeutic agents for angiogenic dependent diseases. To achieve our goal we have chosen the phage display system in order to select for antibodies with angiogenic properties. An antibody phage library has been developed in our laboratory, directed against VEGF molecule, particularly one of it isoforms. The animals were immunized and developed specific antibodies, detected by ELISA and Western-blot. Amplification of the pool of light and heavy chain Ig genes was performed to produce the single chain (ScFv) fragments for library construction. The ScFv antibody display libraries will be then screened in angiogenic settings to isolate antibodies against specific VEGF isoforms and novel cell surface molecular markers expressed by activated human endothelial cells
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Moutel, Sandrine. "Sélection et amélioration d'anticorps recombinants, applications à la recherche fondamentale et à l'immunothérapie." Paris 6, 2009. http://www.theses.fr/2009PA066520.
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Les anticorps (Ac) monoclonaux sont maintenant des outils couramment utilisés aussi bien pour le diagnostic que pour la recherche fondamentale dans l’étude ou la purification des protéines. Avec le développement de l’ingénierie génétique des Ac, il a été possible de cloner des régions variables d’anticorps humains et d’obtenir des banques de grande diversité. Au laboratoire, un investissement particulier a été fait ces dernières années pour adapter les méthodes de sélection d'anticorps recombinants (rAc) aux questions de la biologie cellulaire. La technique du phage display que nous utilisons pour cribler notre banque de scFv, nous a permis d’obtenir de nombreux anticorps notamment contre des échantillons complexes comme des membranes intactes de Golgi ou encore des rAc sensibles aux changements conformationnels indispensables pour nos recherches. Ces scFv ont été sélectionnés rapidement à moindre frais et sans avoir recours au passage par l’animal. Malgré le nombre incroyable de publications décrivant des scFv ou d’autres formats d’rAc, ils n’ont toujours pas réussi à s’imposer comme véritables outils alternatifs aux Ac naturels dans les laboratoires de recherche académiques. Nous avons donc développé notre propre système afin de simplifier et d’améliorer la production d’rAc. Nous avons choisi de les remettre dans un contexte d’Immunoglobuline en leur ajoutant une portion Fc pour les dimériser et de les produire dans des cellules de mammifères. Nous avons choisi le format appelé minibody, il diffère de la structure d’une IgG classique par l’abscence des domaines CH1 et Ckappa, permettant de facilement manipuler l’rAc qui reste sous forme monocaténaire. Pour tous les scFv obtenus au laboratoire, le format minbody devient très robuste. Outre la dimérisation, ce système est versatile, on a pu aisément remplacer le Fc humain par un Fc de lapin ou un Fc de souris. Nous avons aussi développé une nouvelle méthode de sélection d'rAc totalement in vitro. Cette méthode de sélection in vitro d’rAc offre tout d’abord l’avantage d’obtenir des Ac difficiles voire impossibles à sélectionner par immunisation d’animaux , avec des quantités d’Ag de l’orde du µg. L’Ag peut être une protéine très conservée au cours de l’évolution, toxique, être sous une forme native ou dans une conformation d’intérêt. Cette méthode nous a permis de préparer les Ag cibles sans passer par l'expression et la purification chez E. Coli. Deux cribles ont été réalisés avec succès, un contre la GFP pour démontrer la faisabilité du système et un en collaboration avec l’équipe de Philippe Benaroch contre Tsg101. Pour ces études, nous avons développé des méthodes permettant d'obtenir des outils uniques, dont nous étendons maintenant les applications, notamment au domaine thérapeutique. En effet, un avantage lié à ces techniques de sélection in vitro est le fait de disposer simultanément de la séquence nucléotidique codant pour ces rAc entièrement humains. Dans le cadre d’un travail collaboratif, nous nous sommes intéressés aux applications thérapeutiques qui peuvent découler de telles molécules. Le but du projet est d’évaluer l’efficacité de notre rAc chimérique anti-Tn en immunothérapie passive in vivo dans des modèles précliniques de souris greffées par des lignées épithéliales tumorales et d’évaluer la potentialité de son utilisation chez l’homme. C’est un très bon marqueur diagnostic en histologie et aussi pronostic car son expression est corrélée avec le grade de la tumeur Dans tous les tissus Tn est masqué, et il est présent sur la membrane cellulaire dans la majorité des carcinomes (90%), ce qui en fait une cible de choix pour l'immunothérapie. .
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Muller, Benjamin. "Création d'une banque de scFv-phages ciblant des protéines hydrophiles ou membranaires." Thesis, Montpellier 1, 2014. http://www.theses.fr/2014MON13511.
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Actuellement, 60% des médicaments sur le marché ont pour cible des protéines membranaires. Toutefois, l'étude de ces protéines membranaires reste un challenge de par leur structure particulière (domaines transmembranaires hydrophobes et domaines extra- et intra-cellulaires hydrophiles), mais également par leur faible expression sur les cellules.L'entreprise Ciloa, dans laquelle j'ai effectué ma thèse, a développé une technologie brevetée, qui permet d'exprimer à la surface des exosomes, des vésicules membranaires de tailles comprises entre 30 et 100nm, des protéines membranaires natives, grâce à un peptide d'adressage, le DCTM. Cette technologie possède de nombreux domaines d'applications, comme le criblage de médicaments, le développement de vaccins ou encore le développement d'anticorps monoclonaux.L'objectif de ma thèse a été, dans un premier temps, de mettre en place l'outil exosomes recombinants grâce à la technologie de Ciloa et dans un deuxième temps, d'utiliser ces outils pour le développement d'anticorps, grâce aux exosomes recombinants.Ainsi, j'ai d'abord mis au point différentes techniques de caractérisation des exosomes recombinants (ELISA), et également participé à la mise en place de différents protocoles de production et de purification, en fonction leur utilisation. Une fois ces outils optimisés, j'ai pu les utiliser pour le développement d'anticorps. J'ai testé en parallèle deux méthodes de production d'anticorps, une méthode classique, l'hybridation lymphocytaire après immunisation de souris BALB/c, et une méthode plus récente, le criblage d'une banque de scFvs par phage display.L'hybridation lymphocytaire a permis la production d'hybridomes, dont les anticorps ont été criblés sur exosomes, par ELISA. Dans le cadre du criblage par phage display, j'ai participé au développement d'une banque de scFvs, basée sur le modèle du 13R4, dont nous avons modifié les longueurs de boucles des différents CDRs, notamment le CDRH3, afin de cibler les épitopes faiblement accessibles des protéines membranaires. Les sélections de scFvs ont été effectuées sur exosomes recombinants, exprimant des protéines membranairesNowadays, more than 60% of marketed drugs target membrane proteins. However, their study still represents a challenge, essentially due to their particular 3D-structure (hydrophobic transmembrane domains and hydrophilic extra- and intra-cellular domains), but also to their low expression level in cells.Ciloa, the start-up company in which I realized my PhD, has developed a patented technology that enables to express native membrane proteins on exosomes, membrane vesicles of 30 to 100nm, using a pilot peptide called DCTM (for Cytosolic Domain of TransMembrane). This technology displays a lot of different applications, in different domains such as drug screening, vaccines development or monoclonal antibodies (mAbs) development.The purpose of my PhD research was, first, to set up the recombinant exosomal tool using Ciloa's innovative technology, and then to use this tool to develop monoclonal antibodies.Thus, at the beginning of my PhD, I set up exosomal characterization technics, such as ELISA, and I also took part in the setup of several production and purification protocols, depending of the use of exosomes. Once these tools had been optimized, I was able to use them to develop mAbs. I tested two methods, one classical, the generation of hybridoma after Balb/c mice immunizations, and a more recent technology, the screening of scFvs library by phage display.Therefore, I obtained hybridoma and was able to screen the derived antibodies by ELISA on exosomes. Concerning the phage display technology, I took part in the development of a new scFvs library, based on the 13R4 scaffold, of which we changed the CDRs lengths, mostly the CDRH3, in order to target epitopes with low accessibility, such as the one of membrane proteins. The library screening was realized on recombinant exosomes
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Japolla, Greice. "Construção e seleção de uma biblioteca combinatorial de anticorpos contra herpesvirus bovino tipo 1." Universidade Federal de Goiás, 2014. http://repositorio.bc.ufg.br/tede/handle/tede/4215.
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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
Bovine herpesvirus type 1 ( BHV - 1 ) is recognized as an important pathogen of economic losses in cattle , these animals causing diseases known as infectious bovine rhinotracheitis ( IBR ) , infectious pustular vulvovaginitis , infectious balanoposthitis and neurological disorders . For effective control of these diseases, the correct diagnosis is necessary, but none of the available tests enables a quick result made the field. Considering this, the aim of this study was to construct a combinatorial antibody library, for it to be used in the future development of new diagnostic approaches . Two breed White Leghorn chickens were immunized with 105.5 TCID50/ml of BoHV - 1, birds were necropsied , their spleens removed for total RNA extraction , cDNA synthesis , amplification of gene fragments encoding the light chain ( VL ) and heavy ( VH ) and production of scFv ( v) fragments. These fragments were cloned into vectors fagomidiais expressed as fusion proteins on filamentous phage and amplified by infection of E. coli. Selection of viral particles ( fused scFv) binding to the BHV -1 ( biopanning ) by six cycles were performed. The affinity of the scFv antibody library BHV -1 observed in ELISA shows that the produced fragments are reactive to HIV, the use of such antibodies in the development of new diagnostic platforms is possible . The sequencing results showed a reduction of variability in comparison to the dot blot previously performed, and a desirable feature of this process , however, it was possible to sequence the clones efficiently, it has been found , therefore, a need to further analyze the shape of results
O herpesvírus bovino tipo 1 (BoHV-1) é reconhecido como um importante patógeno de perdas econômicas em bovinos, causando nestes animais enfermidades conhecidas como Rinotraqueite Infecciosa Bovina (IBR), vulvovaginite pustular infecciosa, balanopostite infecciosa e desordens neurológicas. Para um efetivo controle destas enfermidades, o diagnóstico correto se faz necessário, porém nenhuma dos testes disponíveis possibilita um resultado rápido feito a campo. Considerando isto, o objetivo deste estudo foi construir uma biblioteca combinatorial de anticorpos, para que esta seja futuramente utilizada no desenvolvimento de novas abordagens diagnósticas. Duas galinhas da raça White Leghorn foram imunizadas com 105,5 DICC50/mL de BoHV-1, as aves foram necropsiadas, seus baços retirados para extração de RNA total, síntese de cDNA, amplificação dos fragmentos gênicos codificantes das cadeias leve (VL) e pesada (VH) e produção de fragmentos scF(v). Estes fragmentos foram clonados em vetores fagomidiais, expressos como proteínas de fusão em bacteriófagos filamentosos e amplificados pela infecção de bactérias E.coli. Foi realizada a seleção de partículas virais (scFv fusionados) ligantes ao BoHV-1 (biopanning) através de seis ciclos. A afinidade da biblioteca de anticorpos scFv ao BoHV-1 observada no teste de ELISA mostra que os fragmentos produzidos são reativos ao vírus, sendo possível a utilização destes anticorpos no desenvolvimento de novas plataformas de diagnóstico. Os resultados de sequenciamento mostraram uma diminuição da variabilidade em comparação ao dot blot realizado anteriormente, sendo uma característica desejável neste processo, porém não foi possível sequenciar os clones de modo eficiente, verificando-se, portanto, a necessidade de analisar de forma mais aprofundada os resultados obtidos .
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Zebedee, Zoë Anna-Marie. "Identification of scFv reagents which recognise the human neural cell adhesion molecule expressed upon neuroblastoma cells." Thesis, University of Bristol, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.390796.
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Saxena, Abhishek. "Construction of immune scFv M13 phage display library and isolation of anti-glycan monoclonal antibodies." Thesis, University of Aberdeen, 2013. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=203295.
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Lamort, Anne-Sophie. "Etude fonctionnelle de la MMp - 12 de macrophage en vue de son ciblage thérapeutique dans la broncho-pneumopathie chronique obstructive." Thesis, Tours, 2015. http://www.theses.fr/2015TOUR4043.
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La bronchopneumopathie chronique obstructive ou BPCO est une atteinte des voies respiratoires causée par le tabagisme. Cette maladie pulmonaire chronique et non réversible pour laquelle il n’y pas de traitement curatif se caractérise par une inflammation permanente du tractus respiratoire. Celle-ci est due à l’afflux massif de cellules de l’inflammation, principalement des neutrophiles et macrophages, qui libèrent après activation, de nombreuses protéases actives. Ces protéases vont alors dégrader les protéines de structure comme l’élastine, ce qui va entraîner la dégradation progressive des alvéoles pulmonaires et au final une altération de plus en plus marquée de la fonction respiratoire. Parmi les différentes protéases présentes dans le poumon, la MMP-12 de macrophage, joue un rôle clé dans la physiopathologie de la maladieChronic obstructive pulmonary disease or COPD is a lung disease caused by tobacco smoking. This is a chronic and non reversible disease for which no curative treatment is available yet. Permanent inflammation of the airways is a hallmark of COPD because immune cells such as neutrophils and macrophages are continuously recruited. Once activated, these cells release numerous active proteases which participate to the degradation of structural proteins of the lungs such as elastin, leading to lung emphysema as a consequence of lung alveoli degradation. Among the different proteases found in the lungs, macrophage MMP-12 has been reported to play a key pathogenic role in COPD development
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Cloutier, Sylvain. "Sélection et production de scFv contre la kallicréine humaine hK2 par la technologie du phage display." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp01/MQ33600.pdf.
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Campos, Lucas Benício. "Atividade tóxica da peçonha de Lachesis muta rhombeata e produção de fragmentos de anticorpos humanos (scFv) contra a peçonha bruta." Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/17/17131/tde-21072016-110802/.
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O tratamento atual indicado para casos de envenenamentos por peçonhas é a administração intravenosa de antivenenos, produzidos através da hiperimunização de animais. Entretanto, os antivenenos disponíveis podem, algumas vezes, não proteger os pacientes e causar reações de hipersensibilidade. Fosfolipases A2 (PLA2), L-aminoácido oxidases (LAAO), metalo e serinoproteases são os principais componentes de peçonhas ofídicas e contribuem para a neurotoxicidade, hemorragia, hemólise, miotoxicidade, cardiotoxicidade e formação de edemas. Foram empregados ensaios para avaliar as atividades das enzimas presentes na peçonha de serpentes da espécie Lachesis muta rhombeata e aquele para atividade de protease foi otimizado. A tecnologia de Phage display foi empregada para a seleção de fagos-anticorpos capazes de reconhecer a peçonha bruta. Os fagos foram amplificados em Escherichia coli TG1 e usados para infectar E. coli HB2151, a qual produz fragmentos de anticorpos humanos solúveis. Estes foram purificados e utilizados em testes de inibição de alguns dos componentes tóxicos da peçonha. Os testes de atividade para PLA2, protease e Laminoácido oxidase foram padronizados com sucesso e as 3 proteínas mostraram elevada atividade enzimática. Após otimização, a quantidade de peçonha necessária para o ensaio de protease foi reduzida em 25 vezes. A massa molecular de PLA2 foi estimada em 17 kDa e as massas moleculares de proteases foram estimadas em 40, 35 e 24 kDa, através de zimogramas. O método de bio panning foi eficiente para a seleção de fagos-anticorpos contra a peçonha bruta. Diversos fragmentos de anticorpos foram purificados e incubados com a peçonha bruta para testar suas capacidades de neutralização sobre cada enzima. Cinco clones demonstraram-se hábeis em inibir a PLA2 através da inibição da hemólise. O clone 4E inibiu 100% da hemólise durante as duas horas de ensaio quando pré-incubado na proporção 2:1 (scFv:peçonha). Os clones 2C e 4E inibiram 100% durante uma hora quando pré-incubados na proporção 1:1 e os clones 2F e 9F inibiram a hemólise parcialmente. Outros testes serão conduzidos para a seleção de clones capazes de neutralizar as demais enzimas, os quais, juntamente com os clones já selecionados, serão analisados através de ensaios in vivo. Espera-se que eles possam contribuir para a construção de um novo antiveneno capaz de superar algumas das dificuldades associadas às técnicas de imunoterapia convencionaisThe current treatment for animal envenoming is the intravenous administration of antivenoms, produced by animal hyperimmunization. Unfortunately, available antivenoms sometimes do not protect patients and may cause hypersensitivity reactions. Phospholipases A2 (PLA2), L-amino acid oxidases, metallo and serine proteases are considered the most important snake venom components and contribute to neurotoxicity, hemorrhage, hemolysis, myotoxicity, edematogeny and cardiotoxicity. Assays for evaluating the enzymes present in Lachesis muta rhombeata venom were developed and the protease one was optimized. Phage display technology was used to select phage antibodies able to recognize the crude venom. Phages were amplified in Escherichia coli TG1 and used to infect E. coli HB2151, which produces human antibody fragments. Inhibition tests aiming the neutralization of some toxic components of the venom were performed using purified antibody fragments. Activity assays for evaluating PLA2, protease and L-aminoacid oxidase were successfully performed and all enzymes showed high activity levels. The molecular mass of PLA2 was estimated in 17 kDa and the molecular mass of proteases were estimated in 40, 35 and 24 kDa, by zymography. After optimizing the conditions for proteolytic assay, it was possible to use 25 times less venom than it was necessary at first. The bio panning method was efficient for selecting specific phage antibodies against the crude venom. Several clones were selected to infect HB2151 and to produce soluble antibody fragments, which were purified and incubated with the venom to test their inhibition capacity over each enzyme. Five clones demonstrated ability to neutralize PLA2 by inhibiting hemolysis. The clone 4E could inhibit 100% of hemolysis for over 2 hours when preincubated at the ratio 2:1 (scFv:venom). Clones 2C and 4E could inhibit 100% for 1 hour when preincubated at the ratio 1:1 and clones 2F e 9F could inhibit partially. Other tests will be performed to select clones able to neutralize other enzymes and, together with the clones already selected, will be evaluated by in vivo experiments. It is expected that they may contribute to the construction of a potential new antivenom able to overcome some of the problems associated with conventional immunotherapy
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Figueiredo, Andreza Soriano [UNESP]. "Clonagem e expressão de fragmentos de anticorpos (scFV) contra o vírus da leucemia felina (FeLV) por phage display." Universidade Estadual Paulista (UNESP), 2010. http://hdl.handle.net/11449/106027.
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figueiredo_as_dr_botfmvz.pdf: 1094123 bytes, checksum: 04a46005f550990f7c07cdcb751f14b5 (MD5)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
O vírus da leucemia felina (FeLV) é um retrovírus que infecta principalmente gatos jovens. Em aglomerados de animais, a infecção pelo FeLV é a que mais contribui para a mortalidade. O emprego de técnicas moleculares de detecção viral permitiu avanços no que diz respeito à caracterização da patogenia e resposta à vacinação. Baseando-se nesses novos resultados, o diagnóstico da infecção deve ser realizado, primeiramente, com um teste de triagem de detecção da proteína de capsídeo p27, e, posteriormente, a confirmação com teste de detecção de DNA proviral. O diagnóstico e separação de animais positivos constituem o mecanismo primordial para conter a disseminação do FeLV. Diante disso, é de grande importância facilitar o acesso e baratear o diagnóstico. A construção de um teste de detecção da p27 baseia-se na produção de anticorpos monoclonais. A técnica de hibridomas é menos prática e demanda mais tempo para a obtenção de resultados satisfatórios quando comparada à técnica de Phage Display. Esta está em franco desenvolvimento e tem ganhado grande aplicabilidade na medicina veterinária. Empregamos o sistema de Phage Display desenvolvido por Krebber e colaboradores (1997). Primeiramente, foi construída uma biblioteca imune em camundongos, em seguida, foram amplificadas as regiões gênicas variáveis das cadeias leve (VL) e pesada (VH) e ligadas com um Linker de (Gli4Ser)4. Esses fragmentos geneticamente construídos derivados de anticorpos são denominados de single chain variable fragment ou scFv. Os scFvs foram fusionados à pIII e apresentados na superfície de fagos filamentos. Após três ciclos de seleção e enriquecimento contra a p27 recombinante produzida no laboratório, onze scFvs foram selecionados e caracterizados com relação à constituição nucleotídica e de aminoácidos. Dentre eles, o scFv 9 e scFv 70 foram escolhidos...
The feline leukemia virus (FeLV) is a retrovirus that infects primarily young cats. In animal clusters, FeLV infection is the largest contributor to mortality. The use of molecular techniques for viral detection has allowed advances with regard to the pathogenicity and response to vaccination. Based on these new findings, the diagnosis of infection should be performed first, with a screening test for detection of p27 capsid protein, and subsequently confirmed with testing for proviral DNA. The diagnosis and segregation of positive animals is the primary mechanism to contain the FeLV spread. Therefore, it is of great importance to facilitate access and lower the diagnosis. The construction of a test for p27 detection relies on monoclonal antibody development. The hybridoma technique is less practical and more time consuming to obtain satisfactory results when compared to Phage Display technology. The latter has been improved rapidly and has gained wide application in veterinary medicine. We employed the Phage Display system developed by Krebber et al. (1997). First, an immune library was built in mice and the variable region of the light and the heavy genes were amplified and connected by a linker of (Gly4Ser)4. This genetically engineered antibody fragments are called single chain variable fragments or scFv. The scFvs were fused to the pIII protein and displayed on the surface of filamentous phages. After three rounds of selection and enrichment against recombinant p27 (produced in the laboratory), eleven scFvs were selected and characterized with respect to nucleotide and aminoacid composition. Among them, scFv 9 and scFv 70 were chosen for subcloning and expression in prokaryotic system for production of heterologous proteins. The scFvs in soluble forms were evaluated for their binding capacity to p27. The scFvs will be employed to the development of an immunoassay for FeLV detect... (Complete abstract click electronic access below)
Books on the topic "Phage displayed scFv"
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Nguyen, Hai Phu. Combination of hu-PBL-SCID mice and scFv phage display library: An effective alternative for hu-mAb production. 2001.
Find full textBook chapters on the topic "Phage displayed scFv"
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Brockmann, Eeva-Christine. "Selection of Stable scFv Antibodies by Phage Display." In Antibody Engineering, 123–44. Totowa, NJ: Humana Press, 2012. http://dx.doi.org/10.1007/978-1-61779-974-7_7.
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Damasceno, Leonardo M., Frank Lee, Gerd Ritter, Lloyd Old, and Carl Batt. "High-Level Expression of a Phage Display-Derived scFv in Pichia pastoris." In Methods in Molecular Biology, 225–36. Totowa, NJ: Humana Press, 2009. http://dx.doi.org/10.1007/978-1-60327-302-2_18.
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Singh, Pawan Kumar, Ranu Agrawal, D. V. Kamboj, and Lokendra Singh. "Construction of Recombinant Single Chain Variable Fragment (ScFv) Antibody Against Superantigen for Immunodetection Using Antibody Phage Display Technology." In Superantigens, 207–25. New York, NY: Springer New York, 2015. http://dx.doi.org/10.1007/978-1-4939-3344-0_17.
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Nahary, Limor, Alla Trahtenherts, and Itai Benhar. "Isolation of scFvs that Inhibit the NS3 Protease of Hepatitis C Virus by a Combination of Phage Display and a Bacterial Genetic Screen." In Methods in Molecular Biology, 115–32. Totowa, NJ: Humana Press, 2009. http://dx.doi.org/10.1007/978-1-60327-302-2_9.
Full textConference papers on the topic "Phage displayed scFv"
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Pruksametanan, Natcha, Montarop Yamabhai, and Pakamatz Khawplod. "Selection of single chain human monoclonal antibody (scFv) against Rabies virus by phage display technology." In 2012 IEEE 6th International Conference on Nano/Molecular Medicine and Engnieering (NANOMED). IEEE, 2012. http://dx.doi.org/10.1109/nanomed.2012.6509127.
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Bezerra, Marcus, Andrea Maranhao, Igor Studart, Larissa Pontes, Marcela Fonseca, and Gilvan Furtado. "Construction of a scFv library using directed evolution for rituximab-based therapies: using phage display towards antibody affinity maturation." In IV International Symposium on Immunobiologicals & VII Seminário Anual Científico e Tecnológico. Instituto de Tecnologia em Imunobiológicos, 2019. http://dx.doi.org/10.35259/isi.sact.2019_32708.
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Figueiredo, Alexandre, Thiago Chaves, Fernando Conte, Rodrigo Silva, Milena Carvalho, Manoela Martins, Adriana Soares, Sheila Lima, and Patrícia Neves. "Development of Single-Chain Variable Fragment (ScFv) antibody against COVID-19 by phage display as a possible tool to diagnostic and treatment." In International Symposium on Immunobiological. Instituto de Tecnologia em Imunobiológicos, 2021. http://dx.doi.org/10.35259/isi.2021_46572.
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Ahmed, Kafil, Vyankatesh Pidiyar, Syed Ahmed, and Sanket Shah. "Development of anti-SARS-CoV-2 specific scFv antibody library from convalescent plasma of COVID-19 recovered patients using phage display technology." In International Symposium on Immunobiological. Instituto de Tecnologia em Imunobiológicos, 2021. http://dx.doi.org/10.35259/isi.2021_46568.
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