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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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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)
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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." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2001. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/NQ58617.pdf.
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Figueiredo, Andreza Soriano. "Clonagem e expressão de fragmentos de anticorpos (scFV) contra o vírus da leucemia felina (FeLV) por phage display /." Botucatu : [s.n.], 2010. http://hdl.handle.net/11449/106027.
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Orientador: João Pessoa Araújo JúniorBanca: Camilo Bulla
Banca: Paulo Eduardo Martins Ribolla
Banca: Julio Lopes Sequeira
Banca: Alexandre Secorum Borges
Resumo: 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... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: 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)
Doutor
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Ribeiro, Vanessa da Silva. "Seleção, caracterização e aplicação de anticorpos scFv (single chain variable fragment) na captura de antígenos para o sorodiagnóstico da neurocisticercose humana." Universidade Federal de Uberlândia, 2012. https://repositorio.ufu.br/handle/123456789/16574.
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Human neurocysticercosis (NC) is an important but neglected cause of epilepsy in
developing countries where the parasite occurs. Expression of single-chain variable fragment
(scFv) antibodies on the surface of bacteriophage is widely used to prepare antibodies with
pre-defined specificities. A phage antibody library was selected against peptides displayed
on phages coupled to beads and total saline extract of Taenia solium metacestodes
immobilized on microtiter plate wells. After two rounds of selection 96 phage clones of each
panning were selected, tested for scFv expression and specificity to each target. Specific
clones were further analyzed by ELISA (Enzyme-linked immunosorbent assay), Dot-blot,
sequencing and immunofluorescence. After selection, three clones were used for antigen
capture to characterize its targets for future immunodiagnostic assays development. Total
saline extract was fractionated on ion exchange resin diethylaminoethyl (DEAE), and
fractions were tested by ELISA to detect sera IgG from: NC, other parasites and health
controls (40 each). The fractions with best diagnostic parameters (sensitivity, specificity,
area under curve and likelihood ratio, calculated by TG-ROC) were selected and subjected to
antigen capture using each purified scFv clone. Each captured fraction was tested by ELISA
to detect IgG in 30 serum samples from each group. In immunofluorescence tests, no
fluorescence was observed in negative controls, and all clones showed a non-uniform
staining profile, and their targets were elucidated through mass spectrometry. After ion
exchange fractionation and ELISA tests, DEAE S2 fraction showed to be the best one and
was used to capture new antigens. DEAE S2 showed 93.3% specificity. Among all clones,
A4 and B6 captured antigens from saline extract and DEAE S2 fraction, respectively, with
the best diagnostic parameters. In conclusion, antibody phage display technology is a
potential approach for the study of antigen-antibody interactions, which can be used to
further elucidate the biology of interaction on neurocysticercosis and to capture new antigens
with potential applications in NC diagnosis and therapeutics.A neurocistocercose humana (NC) é uma doença muito importante, porém negligenciada e é a maior causa de epilepsia em países em desenvolvimento onde a parasitose ocorre. A expressão de fragmentos de cadeia única das regiões variáveis de anticorpo (scFv) na superfície de bacteriófagos é amplamente utilizada para obter anticorpos com especificidades pré-definidas. Uma biblioteca de anticorpos foi utilizada para a seleção de clones específicos à peptídeos expostos em fagos acoplados a beads e ao extrato salino total de Taenia solium (S) imobilizado em placas de microtitulação. Após dois ciclos de seleção, 96 clones de anticorpos foram selecionados contra cada alvo, testados para expressão do scFv e especificidade pelo alvo. Aqueles clones que se mostraram específicos foram melhor analisados por ELISA (Enzyme linked immunosorbent assay), Dot blot, sequenciamento e imunofluorescência. Três clones foram selecionados para serem utilizados na captura antigênica e caracterização do antígeno verdadeiro e para captura de novos antígenos com potencial aplicação em testes diagnósticos. O extrato S foi fracionado em resina de troca iônica diethylaminoethyl (DEAE) para obter frações que foram posteriormente testadas por ELISA para detectar IgG em amostras de soro de pacientes: com NC, outras parasitoses e saudáveis, 40 amostras cada grupo. A fração com melhores parâmetros diagnósticos (sensibilidade, especificidade, área sob a curva e likelihood ratio, calculadas por TG-ROC) foi selecionada e sujeita à captura antigênica usando cada clone de scFv purificado. Cada fração capturada foi testada por ELISA para detectar IgG em 30 amostras de soro de cada grupo. Nos testes de imunofluorescência, nenhuma fluorescência foi observada com os controles negativos e todos os clones mostraram um padrão de marcação não uniforme, seus antígenos alvo foram elucidados por espectrometria de massas. Após fracionamento por troca iônica e ELISA, a fração DEAE S2 se mostrou a melhor e foi utilizada para a captura de novos antígenos. A fração DEAE S2 mostrou especificidade de 93,3%. Dentre todos os clones, o A4 e o B6 capturaram antígenos do extrato S e fração DEAE S2, respectivamente, com os melhores parâmetros diagnósticos. Em conclusão a tecnologia de exposição de anticorpos em fagos é uma técnica potencial para o estudo de interações antígeno-anticorpo utilizadas para melhor elucidar a a biologia da interação na NC e para capturar novos antígenos potencialmente aplicáveis para o diagnóstico da NC.
Doutor em Imunologia e Parasitologia Aplicadas
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Deramchia, Kamel. "Interrogation de la plaque d'athérome par phage-‐display in vivo : une approche pour un ciblage moléculaire à l'aide d'anticorps humains armés pour l'imagerie et la thérapie." Thesis, Bordeaux 2, 2010. http://www.theses.fr/2010BOR21806/document.
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L’athérosclérose est une maladie inflammatoire complexe qui résulte dans la formation de plaques d’athérome à risque de rupture. Le concept récent que ce risque soit lié au contenu de la plaque et non à sa taille se traduit par un nouvel impératif dans le domaine de l’imagerie moléculaire et de la thérapie ciblée. Aujourd’hui l’avènement des approches protéomiques et de criblage à haut débit de banques combinatoires permet l’identification à la fois de nouvelles cibles et d’agents bioactifs. Notre objectif consiste à identifier in situ chez des modèles animaux malades, des fragments d’anticorps capables de cibler spécifiquement les plaques vulnérables d’athérome par phage display in vivo. Ces anticorps seront à la base de nouveaux formats moléculaires pour des études de diagnostic et thérapeutiqueAtheroclerosis is a chronic and progressive inflammatory artery disease. These arteries have morphologically raised lesions: the atherosclerotic plaque. The early atherogenesis is characterized by formation of a lipid-rich core constituted by lipid-laden macrophages. These changes can thin the fibrous cap and render the plaque susceptible to rupture and thrombosis, and eventually if the thrombus occludes the vessels to an acute myocardial infarction. So, there still remains a great need to develop novel diagnosis and therapeutic tools to assess these molecular changes. We have applied a novel in vivo selection scheme to select Antibody-ligands that selectively home into atherosclerotic lesions induced in animal models. Such tissue-specific homing ligands may lead to the characterization of new up-regulated lesion-associated markers and open the way to diagnostic and therapeutic strategies based on non-invasive molecular imaging and selective drug delivery for early lesion detection
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Batista, Thiago Neves [UNESP]. "Clonagem e expressão de fragmentos funcionais(scFv) de anticorpo contra o Parvovírus canino-2 com a técnica da Phage display." Universidade Estadual Paulista (UNESP), 2008. http://hdl.handle.net/11449/103780.
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batista_tn_dr_botfmvz_prot.pdf: 1218510 bytes, checksum: 40011e3f4a7d38b02ece0fb1208465c5 (MD5)Universidade Estadual Paulista (UNESP)
A parvovirose canina, uma das principais doenças infecto-contagiosas que acomete cães, é causada pelo Parvovírus canino-2 (CPV-2). Desde sua ' escrição importantes mutações ocorreram originando três tipos: CPV-2a e 2b, comumente detectados no Brasil, e o CPV-2c. Essas alterações antígênicas caracterizam o CPV como um dos principais modelos de evolução viral, sendo etectáveis com painéis de anticorpos monoclonais em técnicas simples como hemaglutinação e neutralização in vitro. A tecnologia de apresentação de :J oteínas em bacteriófagos (Phage display) tem diversas aplicações, dentre elas a apresentação de fragmentos recombinantes de anticorpos funcionais scFv), que podem ser produzidos em grande quantidade. Este trabalho utilizou Phage Display para expressar fragmentos scFv contra o CPV-2 , com uma etodologia descrita (Krebber, 1997). Após o cultivo e purificação do vírus, camundongos da linhagem High seleção IV A foram imunizados com o CPV-2 , sendo em seguida extraído RNA total do baço. A amplificação das cadeias leve e pesada, e sua ligação por SOE-PCR, permitiu a clonagem do scFv no fagomídeo pAK 100. Após a transformação da E.coli XL-1 blue, bacteriófagos 13 foram expressos apresentando o fragmento scFv. Três processos de seleção foram realizados, e após '0 Phage-ELlSA, um grupo de fagos foi selecionado. A partir deste um screening foi realizado por PCR sendo dois lones detectados. Após nova expressão de ambos uma nova reação de Phage-ELlSA foi realizada e demonstrou a especificidade destes clones com o CPV-2. Até o presente momento esta é a primeira descrição da técnica de Phage display apresentando fragmentos de anticorpo anti-CPV-2.
Canine parvoviral disease, one of the most common infectious disorders of dogs, is caused by canine parvovirus (CPV-2). Several mutations have accurred since it was described in the late 1970s, originating three antigenic pes: CPV-2a and 2b, most commonly found in Brazil, and the CPV-2c. These tigenic differences characterize CPV-2 as an important model of virus evolution, and could be detected by monoclonal antibody panel in hemmaglutination and in vitro neutralization. Phage display has many a plications and have been used displaying functional antibodies fragments scFv) , which can be produced in large amount. The technology described here
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Batista, Thiago Neves. "Clonagem e expressão de fragmentos funcionais(scFv) de anticorpo contra o Parvovírus canino-2 com a técnica da Phage display /." Botucatu : [s.n.], 2008. http://hdl.handle.net/11449/103780.
Full textAbstract:
Resumo: A parvovirose canina, uma das principais doenças infecto-contagiosas que acomete cães, é causada pelo Parvovírus canino-2 (CPV-2). Desde sua '" escrição importantes mutações ocorreram originando três tipos: CPV-2a e 2b, comumente detectados no Brasil, e o CPV-2c. Essas alterações antígênicas caracterizam o CPV como um dos principais modelos de evolução viral, sendo etectáveis com painéis de anticorpos monoclonais em técnicas simples como hemaglutinação e neutralização in vitro. A tecnologia de apresentação de :J oteínas em bacteriófagos (Phage display) tem diversas aplicações, dentre elas a apresentação de fragmentos recombinantes de anticorpos funcionais scFv), que podem ser produzidos em grande quantidade. Este trabalho utilizou Phage Display para expressar fragmentos scFv contra o CPV-2 , com uma etodologia descrita (Krebber, 1997). Após o cultivo e purificação do vírus, camundongos da linhagem High seleção IV A foram imunizados com o CPV-2 , sendo em seguida extraído RNA total do baço. A amplificação das cadeias leve e pesada, e sua ligação por SOE-PCR, permitiu a clonagem do scFv no fagomídeo pAK 100. Após a transformação da E.coli XL-1 blue, bacteriófagos 13 foram expressos apresentando o fragmento scFv. Três processos de seleção foram realizados, e após '0 Phage-ELlSA, um grupo de fagos foi selecionado. A partir deste um screening foi realizado por PCR sendo dois lones detectados. Após nova expressão de ambos uma nova reação de Phage-ELlSA foi realizada e demonstrou a especificidade destes clones com o CPV-2. Até o presente momento esta é a primeira descrição da técnica de Phage display apresentando fragmentos de anticorpo anti-CPV-2.Abstract: Canine parvoviral disease, one of the most common infectious disorders of dogs, is caused by canine parvovirus (CPV-2). Several mutations have accurred since it was described in the late 1970s, originating three antigenic pes: CPV-2a and 2b, most commonly found in Brazil, and the CPV-2c. These tigenic differences characterize CPV-2 as an important model of virus evolution, and could be detected by monoclonal antibody panel in hemmaglutination and in vitro neutralization. Phage display has many a plications and have been used displaying functional antibodies fragments scFv) , which can be produced in large amount. The technology described here :: the expression of antibody recombinant fragments - scFv - against CPV-2, the methodology previously described (Krebber, 1997). After cultivation and purification of virus, High IV A mice were immunized with CPV-2, total RNA s extracted from spleen cells. Light and heavy chains were amplified, and en linked by SOE-PC R; and the product was cloned into a phagemid (pAK ). After the transformation on E. coli XL-1 blue, M13 bacteriophages were - ressed presenting the scFv fragment. Series of 3 rounds of panning was ied out, and after a Phage-ELlSA a group of phage was selected. A PCR - eening was conducted and two clones detected. After new expression a age-ELlSA was performed and demonstrated the specificity of these clones against the CPV-2. Until the moment this is the first description of the use of the :J age display, producing antibody fragments against CPV-2. -
Orientador: João Pessoa Araújo Junior
Coorientador: Paulo Eduardo Martins Ribolla
Banca: Ramon Kaneno
Banca: Alice A. F. Alfieri
Banca: Hélio José Montassier
Banca: Alexandre S. Borges
Doutor
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Ndlovu, Siphumelele. "The isolation of single chain variable region fragments (scFvs) from a phage display library, and expression of the isolated scFvs in Nicotiana benthamiana." Master's thesis, Faculty of Science, 2020. http://hdl.handle.net/11427/32303.
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Monoclonal antibodies (mAbs) are an important tool for both therapeutic and nontherapeutic applications. Their increased demand is due to their ability to recognize and bind specifically to a wide range of antigens. In addition to full-size antibodies, one can also utilise smaller antibody fragments, single chain variable region fragments (scFvs), which like full-size mAbs, are also capable of specific antigen-binding. The constant and rapidly expanding use of antibodies and their derivatives presents a need for a fast and effective method of production. Traditionally, antibodies have been produced using hybridoma technology. They have also been successfully produced in other expression hosts such as bacteria, yeasts, insect cells and mammalian cell lines. However, these expression systems come with a few disadvantages, some of which include high maintenance costs as well as lengthy and laborious production protocols. This dissertation describes the use of phage display technology to screen for and identify scFvs that bind to three different test antigens. Phage display library technology involving the expression and presentation of antibody or antibody derivatives on the coat surfaces of phage particles. It is considered to be a preferable alternative to hybridoma technology because it eliminates the requirement for immunization of animals, making it a more rapid and animal-friendly method for the production of antibodies compared to that of hybridoma technology. A naïve mouse scFv phage display library was screened with appropriate antigens to isolate scFvs which bind to rabbit IgG, human IgG and the Shuni virus (SHUV) N protein. Isolated scFvs were sequenced, cloned and tested for binding to their cognate antigens using phage ELISA, phage dot blots and phage western blots. ScFvs displaying the highest affinities for their respective antigens were selected for cloning and expression in plants, as this expression system is scalable, cheaper, safe and facilitates posttranslational modifications to recombinant proteins such as glycosylation. Rabbit IgG and human IgG scFvs were isolated successfully from the mouse scFv phage library, however, successful binding of the scFvs to the respective antigens by western blotting and ELISAs was not demonstrated. On further investigation, it appeared that the protocols were flawed, as the secondary anti-mouse AP conjugate, iv used in the western blots and ELISAs was found to cross-react with both rabbit and human IgG. Since we were not able to pinpoint scFvs with high binding affinity, the mouse phage display library was screened for scFvs that bound to SHUV N protein instead. This was more successful in that several scFvs with high binding affinity were isolated. Three scFvs with the highest binding affinity for the SHUV N protein were selected and their nucleotide sequences determined. Due to time constraints only 2 of the identified scFvs were selected for further cloning and expression in plants. Both scFvs were cloned into the pTRA-HRPB2SEKDEL plant expression vector that contains the gene sequence for a his6x tag to assist with downstream purification as well as a horse radish peroxidase (HRP) gene. Cloning scFvs into this vector allows their fusion to HRP, resulting in the production of potential reagents for use as secondary antibodies in western blots and ELISAs. The cloned scFvs were expressed transiently in tobacco plants using Agrobacterium-mediated infiltration. Plant expression of the HRP-fused scFvs was optimized; both were optimally expressed at 5 days post infiltration (dpi) when co-expressed with a silencing suppressor (pBIN-NSs). Extraction of the scFvs from the plants was most effective when a bicine buffer with a pH of 8.4 was used. Partial purification of the scFvs was achieved by isoelectric and ammonium sulphate precipitation. Preliminary tests were done to test functionality of the partially purified scFvs, in which the ability of the scFvs to recognize and bind to the SHUV N protein in a dot blot was tested. However, both were found to be non-functional in this regard. Further investigation into the reason for the demonstration of non-functionality showed that the HRP was being spontaneously cleaved from the scFv. This study demonstrates that it is possible to isolate antigen-specific scFvs from a phage display library. However, their binding capacity needs to be analysed fully prior to incorporating them into fusion proteins which can be used as potential diagnostic reagents.
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Fernandes, Camila Cesário [UNESP]. "Produção de fragmentos de anticorpos monoclonais (scFv) contra isolados de campo do vírus da bronquite infecciosa das galinhas utilizando phage display." Universidade Estadual Paulista (UNESP), 2009. http://hdl.handle.net/11449/94946.
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fernandes_cc_me_jabo.pdf: 1349174 bytes, checksum: b0d752648346a29041d0fbd5f330fbf6 (MD5)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Anticorpos monoclonais se constituem na base de vários testes usados na detecção e na identificação de antígenos. Nesse contexto, tais imuno-reagentes têm sido extensivamente empregados na identificação de estirpes virais envolvidas na etiologia de surtos de bronquite infecciosa a campo, permitindo o aperfeiçoamento das técnicas de detecção e caracterização antigênica do vírus da bronquite infecciosa das galinhas (VBI). No presente estudo, uma biblioteca de fragmentos de anticorpos de galinha originalmente preparada por “phage display” contra a estirpe vacinal (H120) do VBI, foi usada para a seleção de fragmentos de anticorpos recombinantes com reatividade cruzada para as estirpes heterólogas IBVPR01, IBVPR05, isoladas de surtos a campo no Brasil e SE-17, isolada nos Estados Unidos. Após três ciclos de “panning”, foi identificado pelo ELISA um conjunto de 15 anticorpos scFv expressos em fagos e com reatividade cruzada para essas mesmas estirpes do VBI. A análise por Western-blotting revelou que três desses clones apresentavam fagos expressando fragmentos de anticorpos monoclonais com reatividade cruzada para a nucleoproteína N das três estirpes do VBI e também para a forma recombinante dessa nucleoproteína derivada da estirpe M41. Concluindo, os fragmentos de anticorpos monoclonais recombinantes scFv-N produzido em fagos interagem com um epítopo mais conservado da proteína N do VBI e apresentam um grande potencial para utilização na detecção e no diagnóstico direto desse vírus e no estudo de evolução de variantes desse vírus.
Monoclonal antibodies are the basis of various techniques used for antigen detection or characterization, and their use is specially recommended for the identification of viral strains involved in the etiology of outbreaks of infectious bronchitis, because these antibodies are homogeneous, highly specific and fully characterized, allowing the improvement of detection of immunological techniques and antigenic characterization of avian infectious bronchitis virus strains (IBV). We used a phage display library prepared previously against the IBV vaccine strain (H120) for the selection of new scFv antibody fragments reacting with heterologous IBV strains isolated from outbreaks in Brazil (IBVPR01, IBVPR05) and USA (SE-17). After three cycles of panning a set of 15 scFv antibodies was expressed in phages and exhibited crossreaction in ELISA with these three viral strains. Western-blotting analysis showed that three of this clone set were expressing scFv specific for the nucleoprotein of these IBV strains, as well as to the recombinant form of this protein derived from M41 strain of IBV. In conclusion, the recombinant fragments of monoclonal antibodies expressed by phage-display technique have a great potential for future use in immunodiagnostic techniques and study the evolution of variant strains of this virus.
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Fernandes, Camila Cesario. "Produção de fragmentos de anticorpos monoclonais (scFv) contra isolados de campo do vírus da bronquite infecciosa das galinhas utilizando phage display /." Jaboticabal : [s.n.], 2009. http://hdl.handle.net/11449/94946.
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Orientador: Hélio José MontassierBanca: Camillo Del Cistia Andrade
Banca: Janete Apparecida Desidério Sena.
Resumo: Anticorpos monoclonais se constituem na base de vários testes usados na detecção e na identificação de antígenos. Nesse contexto, tais imuno-reagentes têm sido extensivamente empregados na identificação de estirpes virais envolvidas na etiologia de surtos de bronquite infecciosa a campo, permitindo o aperfeiçoamento das técnicas de detecção e caracterização antigênica do vírus da bronquite infecciosa das galinhas (VBI). No presente estudo, uma biblioteca de fragmentos de anticorpos de galinha originalmente preparada por "phage display" contra a estirpe vacinal (H120) do VBI, foi usada para a seleção de fragmentos de anticorpos recombinantes com reatividade cruzada para as estirpes heterólogas IBVPR01, IBVPR05, isoladas de surtos a campo no Brasil e SE-17, isolada nos Estados Unidos. Após três ciclos de "panning", foi identificado pelo ELISA um conjunto de 15 anticorpos scFv expressos em fagos e com reatividade cruzada para essas mesmas estirpes do VBI. A análise por Western-blotting revelou que três desses clones apresentavam fagos expressando fragmentos de anticorpos monoclonais com reatividade cruzada para a nucleoproteína N das três estirpes do VBI e também para a forma recombinante dessa nucleoproteína derivada da estirpe M41. Concluindo, os fragmentos de anticorpos monoclonais recombinantes scFv-N produzido em fagos interagem com um epítopo mais conservado da proteína N do VBI e apresentam um grande potencial para utilização na detecção e no diagnóstico direto desse vírus e no estudo de evolução de variantes desse vírus.
Abstract: Monoclonal antibodies are the basis of various techniques used for antigen detection or characterization, and their use is specially recommended for the identification of viral strains involved in the etiology of outbreaks of infectious bronchitis, because these antibodies are homogeneous, highly specific and fully characterized, allowing the improvement of detection of immunological techniques and antigenic characterization of avian infectious bronchitis virus strains (IBV). We used a phage display library prepared previously against the IBV vaccine strain (H120) for the selection of new scFv antibody fragments reacting with heterologous IBV strains isolated from outbreaks in Brazil (IBVPR01, IBVPR05) and USA (SE-17). After three cycles of panning a set of 15 scFv antibodies was expressed in phages and exhibited crossreaction in ELISA with these three viral strains. Western-blotting analysis showed that three of this clone set were expressing scFv specific for the nucleoprotein of these IBV strains, as well as to the recombinant form of this protein derived from M41 strain of IBV. In conclusion, the recombinant fragments of monoclonal antibodies expressed by phage-display technique have a great potential for future use in immunodiagnostic techniques and study the evolution of variant strains of this virus.
Mestre
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Goffinet, Marine. "Conception et obtention d'un anticorps spécifique des formes activées des Rho GTPases." Toulouse 3, 2007. http://thesesups.ups-tlse.fr/64/.
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Les protéines Rho A, B et C dont l'activité est régulée par l'échange GDP/GTP seraient impliquées dans la tumorigenèse. Même si la surexpression dans les tumeurs des protéines Rho est largement démontrée et qu'il est établi que seule la forme activée est capable d'entraîner la transmission des signaux, seules quelques études ont corrélé l'activation des protéines Rho à la gravité des cancers. Nous avons identifié par " phage display " un scFv (C1) capable de distinguer spécifiquement la conformation activée (liée au GTP) des protéines Rho A, B et C. L'anticorps soluble C1 exprimé en fusion avec la partie N-terminale de la protéine pIII du phage (C1-N1N2) s'associe spécifiquement aux protéines Rho activées endogènes des cellules HeLa (immunofluorescence). Cette propriété originale permettra, en association avec les anticorps non conformationnels anti-RhoA et anti-RhoB d'analyser l'activation des Rho dans le développement tumoralThe Rho GTPases A, B and C proteins, members of the Rho family whose activity is regulated by GDP/GTP cycling, function in many cellular pathways controlling proliferation and have recently been implicated in tumorigenesis. Although overexpression of Rho GTPases has been correlated with tumorigenesis, only their GTP-bound forms are able to activate the signalling pathways implicated in tumorigenesis. Thus, the focus of much recent research has been to identify biological tools capable of quantifying the level of cellular GTP-bound Rho, or determining the subcellular location of activation. However useful, these tools used to study the mechanism of Rho activation still have limitations. The aim of the present work was to employ phage display to identify a conformationally-specific single chain fragment variable (scFv) that recognizes the active form of Rho GTPases and is able to discriminate the activated (GTP bound) form of Rho in endogenous settings. After five rounds of phage selection using a constitutively activated mutant of RhoB (RhoBQ63L), three scFvs (A8, C1 and D11) were selected for subsequent analysis. Further biochemical characterization was pursued for the single clone, C1, exhibiting an scFv structure. C1 was selective for the GTP-bound form of RhoA, RhoB, as well as RhoC, and failed to recognize GTP-loaded Rac1 or Cdc42, two other members of the Rho family. Soluble C1 expressed in fusion with the N-terminal of phage protein pIII (scFv C1-N1N2) specifically associated with GTP-loaded recombinant RhoA and RhoB via immunoprecipitation, and endogenous activated Rho in HeLa cells as determined by immunofluorescence. We identified an antibody, C1-N1N2, specific for the GTP-bound form of RhoB from a phage library, and confirmed its specificity towards GTP-bound RhoA and RhoC, as well as RhoB. The success of C1-N1N2 in discriminating activated Rho in immunofluorescence studies implies that this new tool, in collaboration with currently used RhoA and B antibodies, has the potential to analyze Rho activation in cell function and tumor development
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Zhou, Jie. "Generation of scFv recombinant antibodies with the help of the phage display system against the microtubule associated protein Tau and the kinase MARK." [S.l.] : [s.n.], 2000. http://deposit.ddb.de/cgi-bin/dokserv?idn=960264000.
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Koopman, Tammy L. "Production of Porcine Single Chain Variable Fragment (SCFV) selected against a recombinant fragment of Porcine Reproductive and Respiratory Syndrome virus non structural protein 2." Thesis, Kansas State University, 2011. http://hdl.handle.net/2097/13189.
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Master of ScienceDepartment of Diagnostic Medicine/Pathobiology
Richard 'Dick' Hesse
Carol Wyatt
Over the last two decades molecular laboratory techniques have enabled researchers to investigate the infection, replication and pathogenesis of viral disease. In the early eighties, Dr. George Smith developed a unique system of molecular selection. He showed that the fd bacteriophage genome could be manipulated to carry a sequence of DNA coding for a protein not contained in the phage genome. Infection of the recombinant bacteriophage or phagemid into a specific strain of the bacterium, Escherichia coli, produced progeny phage with the coded protein displayed as a fusion with the phage's coat protein. Antibody phage display utilizes the same technology with the DNA encoding an antibody fragment. The DNA insert can carry the information to produce either a single chain variable fragment (scFv) producing the heavy chain variable and light chain variable (VH-VL) portion or a Fab fragment which also contains the heavy chain constant 1 with the light chain constant (CH and CL) portion of an antibody. Screening an antibody phage display library has the possibility of producing an antibody not produced in the normal course of immune selection. This decade also saw the emergence of a viral disease affecting the porcine population. The Porcine Reproductive and Respiratory Syndrome virus (PRRSV) has been one of the most costly diseases affecting the pig producer. Molecular investigations found that PRRSV is a single, positive-stranded RNA virus which codes for five structural and 12-13 nonstructural proteins producing an enveloped, icosahedral virus. An interesting characteristic of PRRSV is the ability to produce infective progeny with genomic deletions, insertions and mutations within the nonstructural protein 2 (nsp2). With this knowledge, many researchers have produced marker vaccines containing fluorescent tags with the hope of developing a DIVA (Differentiate Infected from Vaccinated Animals) vaccine. In my Master‟s studies, I studied the techniques of antibody phage display technology and how to apply these methods to producing scFvs which recognize a recombinant PRRSV nsp2 fragment protein and the native protein during infection of MARC-145 cells.
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Burden, Matthew. "An investigation into making a bispecific scFv antibody that recognises both pIgR. and IgG Fc by phage display technology in order to transport IgG to mucosal surfaces." Thesis, University of Bristol, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.486114.
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The polymeric immunoglobulin receptor (pIgR) transports polymeric immunoglobulins across the epithelial cell layer into mucosal secretions. It was hoped that pIgR could be exploited to actively transport IgG by constructing a bispecific· molecule capable of simultaneously binding IgG and pIgR. Increasing the IgG' delivered to mucosal secretions may benefit patients undergoing intravenous immunoglobulin therapy by increasing the mucosal defence against invading pathogens. Phage display, using synthetic phagemid libraries (I and J), was used to generate scFv to secretory component (SC), the extracellular portion of pIgR. Phage raised against peptides of pIgR yielded no SC binding clones. Subsequent selections against SC were successful. An in vitro transport assay was used to measure transport of anti-SC clones. No transport could be detected in the initial assay, so it was optimised to increase sensitivity. Subsequent selections using this assay suggested that the dominant epitope recognised by the J library in SC was distinct from those that bound on pIgR that led to the highest level of transport. The dominant epitope recognised by the I library was present on both pIgR and SC. The clones were of weak binding possibly because of the restrictive design ofthe library and apparent bias seen in the sequences ofthe unselected clones. Affinity maturation was applied to the VL CDR3 in an attempt to improve the level of transcytosis of an anti-SC clone in the transport assay. A small improvement of transport was observed. Recombinant techniques used to create a bispecific scFv tandem were unsuocessful, in that its construction led to loss of binding specificities of both scFvs. Investigations in vivo were carried out as the first step of establishing an assay to test the final bispecific reagent. Study of the transport of an anti-pIgR phage suggested it was enriched compared to control phage in the faeces of mice injected intravenously.
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Chaaya, Nancy. "Anticorps catalytiques et répertoires immuns murins : analyse génétique, biochimique et bio-informatique." Thesis, Compiègne, 2019. http://www.theses.fr/2019COMP2495.
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A la fin des années 80, des anticorps catalytiques ont été découverts dans le sérum de patients, en particulier de patients atteints de maladies auto-immunes. Certains anticorps catalytiques ont un effet bénéfique sur la santé, tandis que d'autres sont délétères. Afin de comprendre le lien existant entre anticorps catalytiques et pathologies auto-immunes, des travaux antérieurs ont mené à la synthèse de quatre banques de fragments d’anticorps (scFv) exposés en surface de phages, représentant différents fonds génétiques et états immunologiques. Les scFv, constitues des régions variables des chaines lourdes (H) et légères (L) des anticorps, sont codes par différents segments de gènes d'immunoglobuline : V-D- J pour la chaine lourde, V et J pour la chaine légère. Dans l'objectif de récolter des informations sur l’immunogénétique des anticorps catalytiques, la distribution des sous-groupes de gènes au sein de chaque répertoire a été étudiée, en se basant sur l’étude de plus de 300 000 séquences. L'analyse des données NGS a montré une expression différentielle des sous-groupes de gènes selon la banque d’origine, suggérant que le fond génétique et / ou l'état immunologique influencent l'expression du sous-groupe de gènes d'immunoglobuline. La présence d'anticorps potentiellement catalytiques à activité β-lactamase a ensuite été étudiée dans les quatre banques par une approche in silico de modélisation tridimensionnelle. Les résultats suggèrent que certaines banques expriment potentiellement plus d'anticorps catalytiques que d'autres. Enfin, dans le but de valider cette approche in silico, une approche in vitro a été initiée. Cinq scFv exposés à la surface des phages ont été sélectionnés lors d'un travail précèdent par un processus itératif sur la base de leur activité catalytique. Chacun possède une structure primaire et tertiaire unique. L’un d’entre eux, le scFv P90C2, a été cloné et exprimé dans des bactéries E. coli BL21 (DE3) sous forme de corps d'inclusion, puis solubilise et enfin renaturé. Bien que le scFv P90C2 soluble conserve son activité de reconnaissance, son pouvoir catalytique est complètement perdu. L’influence de différents paramètres sur la fonctionnalité du scFv a été évaluée : (i) optimisation des conditions du protocole de renaturation, (ii) choix des codons à l’origine de la séquence peptidique du scFv, et enfin (iii) influence de la protéine de fusion pIIIIn the late 80s, catalytic antibodies have been discovered in the serum of patients, especially patients with auto-immune diseases. Some of the catalytic antibodies appear to have a beneficial effect on health while others are deleterious. In order to understand the link between catalytic antibodies and immune system pathologies, previous work leaded to 4 single chain Fragment variable (scFv) libraries exposed on phage surface, representing different genetic backgrounds and immunological states. The scFvs, composed with the variable regions of the heavy (H) and light (L) chains, are encoded by immunoglobulin gene subgroups V(H), D(H), J(H), V(L) and J(L). With the objective to decipher the potential origin of catalytic antibodies, a statistical representation of each subgroup within each repertoire has been done, based on more than 300 000 sequences. The NGS data analysis showed a variable expression of some gene subgroups (comprising “rare” ones) between the 4 libraries showing that the genetic background and/or the immunological state influence immunoglobulin gene subgroup expression. Then, we investigated the presence of antibodies with potent active sites in the libraries by molecular modelling. Libraries express more putative catalytic antibodies than others depending on the genetic background and the immunological state profile. Finally, in the objective to validate this in silico approach, an in vitro approach was considered. 5 scFvs exposed on phage surface have thus been selected during a previous work by iterative process on the basis of their catalytic activity: β-lactamase like activity. Each of them displays a unique primary and tertiary structure. The scFvs exposed on the phage surface must be catalytically active while expressed in soluble form too. One of the selected scFvs, P90C2, was optimized and expressed in E. coli BL21 (DE3) bacteria in the form of inclusion bodies and then solubilized and refolded. Although soluble P90C2 fully retained its binding activity, its catalytic potency was completely lost. Further experiments aimed to i) optimize refolding protocol, ii) study the impact of scFv codon-optimization, and iii) show the influence of the pIII fusion protein on the scFv catalytic activity
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Siepert, Eva-Maria [Verfasser]. "Towards the generation of recombinant and pharmaceutically relevant target proteins : phage display-based isolation of specific scFv antibodies against metastasizing pancreatic carcinoma for clinical application and development of novel fluorescent reporter tags for on-line monitoring during target protein production / Eva-Maria Siepert." Aachen : Hochschulbibliothek der Rheinisch-Westfälischen Technischen Hochschule Aachen, 2014. http://d-nb.info/104867049X/34.
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Shahsavarian, Melody. "Genesis of immune diversity and selection of catalytic antibodies : a new investigation." Thesis, Compiègne, 2015. http://www.theses.fr/2015COMP2215/document.
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Les anticorps catalytiques (ou abzyme) ont fait l’objet de nombreuses recherches et ont été produits pour réaliser de nombreuses réactions. Ces protéines ont été ensuite découvertes dans le sérum d’individus sains ou atteints de pathologies, dont les pathologies autoimmunes. Les études suggèrent que ces abzymes peuvent avoir des effets bénéfiques ou délétères sur la santé des individus. L’origine des anticorps catalytiques et leur rôle restent ambigus et doivent être approfondis. Nous avons développé une nouvelle stratégie visant à étudier les abzymes, basée sur la technologie du phage display. Nous avons construit 4 banques de fragments d’anticorps, chacune présentant un répertoire immun différent (fond génétique et état d’immunitaire) : saine et naïve, saine et immunisée, autoimmune et naïve, et autoimmune et immunisée. Les stratégies d’amplification et de clonage des régions variables des immunoglobulines ont été conçues afin d’optimiser la taille et la diversité des banques. Nous avons rassemblé les 4 banques en une banque unique élargie contenant 2.7×109 séquences. L’analyse des séquences a mis en évidence des différences dans les profils d’expression des sous-groupes de gènes selon la banque. Nous avons ensuite procédé à la sélection d’abzymes à activité β-lactamase en utilisant deux cibles : un peptide cyclique, et un dérivé de sulfone pénam, inhibiteurs de l’enzyme. Nous avons sélectionné 5 abzymes. Chacun de ces immunoglobulines ont des séquences protéiques propres, incluant un potentiel site actif. Ces résultats montrent que différents motifs peuvent assurer la fonction catalytique de la β-lactamase, confirmant la flexibilité moléculaire de cette enzymeCatalytic antibodies (or abzymes) have been the focus numerous studies for some decades and have been produced with the ability to catalyze a wide range of reactions. They have also been discovered naturally in normal physiological and pathological conditions, notably on the background of autoimmune disease. Some have beneficial effects and others are detrimental to individual’s health. Hence, the origin of abzymes and their role in the immune response are ambiguous and must be enhanced. We have developed a novel strategy for the study of abzymes based on the phage display technology. We have constructed 4 libraries representing 4 murine immune repertoires with different genetic backgrounds and immunological states : healthy and naïve, healthy and immunized, autoimmune and naïve , and autoimmune and immunized. The strategies for the amplification and cloning of the immunoglobulin (lg) variable regions have been designed to optimize the size and diversity of the libraries. We have been able to pool the four libraries to create a large repertoire of size 2.7x109. After sequence analysis, we have found a number of statistically significant differences between the libraries. We have then used two strategically chosen targets to select for antibodies endowed with β lactamase activity : a cycle peptide and a penam sulfone, both inhibitors of the enzyme. We have selected for a total of 5 lgs endowed with β lactamase activity. The selected abzymes have different amino acid sequences. 3D modeling has provides insights on potential active sites demonstrating the ability of different structures to maintain the β lactamase activity and confirming the flexibility of the active site
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Chou, Ying-Hsiu, and 周盈秀. "Construction of human phage-displayed scFv library." Thesis, 2009. http://ndltd.ncl.edu.tw/handle/13301253686668832243.
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碩士國立成功大學
醫學檢驗生物技術學系碩博士班
97
Antibodies are applied not only for diagnosis, but also in research and clinical treatments. In order to generate high-affinity monoclonal antibodies, three techniques have been developed including hybridoma engineering, transgenic mice and phage-displayed antibody libraries. Although the hybridoma technology is the major tool for monoclonal antibody production, it still has some problems. For example, the methodology cannot be curable directly in human disease. Hence, the phage antibody display system has become more attractive to scientists. We attempted to develop a na��ve human single chain antibody (scFv) library by phage display system. We first extracted total RNA from the leukocytes of peripheral blood from 290 volunteers. The heavy chain and light chain cDNA fragments were synthesized by reverse transcription reaction. Thirty-one forward and reverse primers were used for the cloning of heavy chain variable regions (VH) and light chain variable regions (VL) fragments from the cDNA fragments. The cloned heavy and light chain variable regions were amplified and linked with linkers by overlapping PCR. The linkers encoded the 15 amino acids (Gly4Ser)3 that have overhangs of perfect complementarity with VH and VL genes. The purified scFv fragments were ligated to the phagemid pIGT5 for electroporation into Escherichia coli HB2151 competent cells. In order to increase the binding avidity of scFv during bio-paining, we had constructed pIGT5 phagemid which expressed trypsin and enterokinase cutting site between pIII and scFv. We also established Ex-phage in which the pIII protein could not be synthesized in non-amber suppressed strain owing to the amber codon before pIII. Hence, the binding phage can be released after enzymatic digestion and amplified for next paining. After the library constructed, we will apply this antibody library to discover new carbohydrate antibodies.
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Huang, Shih-Tsung, and 黃仕聰. "Development of Fully Human Anti-HDGF Antibody from Phage-Displayed Human Naïve ScFv Library." Thesis, 2019. http://ndltd.ncl.edu.tw/handle/92wr6a.
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博士國立中山大學
海洋生物科技博士學位學程
107
Hepatocellular carcinoma (HCC), a deadly disease worldwide. For advanced HCC therapy, Sorafenib is the only conventional drug, however, low response rate, drug resistance, and strong side effects had been reported in Sorafenib therapy. Therefore, investigation of novel therapeutic strategy is a critical issue. HDGF is a cancerous factor in various cancer types including HCC. To develop anti-HDGF neutralizing antibody, phage display technology provides an effective in vitro selection strategy to identify fully human antibody. In the biopanning, thirteen HDGF affinity phage clones had been identified from phage display human naïve scFv library using unique Fc fusion protein/protein G dynabeads method. After fully human IgG construction and production, these antibodies were further confirmed binding affinity using ELISA, immunofluorescence, and HDGF conditional knockdown Huh7 cell line. Among them, hmAb-7 exhibited multiple anti-cancer activity, including cell growth, colony formation, invasion and sphere formation in Huh7, HepG2, and SK-Hep1 cells. The anti-cancer effects of hmAb-7 might participated with downregulation of PI3K/AKT/mTOR signaling pathway. The hmAb-7 inhibits of invasion through MMP-2 down-regulation; sphere formation was eliminated through downregulation of cancer stemness genes. In continuously cultured Huh7 and HepG2 cells, these cells present higher colony and spheroid forming features, however, the cancerous behavior able to abolish through hmAb-7 treatment. In sorafenib resistant Huh7 cells, hmAb-7 presents colony and sphere inhibition ability. Furthermore, the Huh7 xenograft model in NOD/SCID mice was performed and the data indicated that hmAb-7 slightly inhibited tumor growth (p = 0.1091) and prolong the overall survival for 14 days. Although anti-HDGF hmAb-7 single treatments not significantly inhibit tumor growth in vivo, but based on the cancerous inhibition effects in vitro, combination therapy might provide an approach to enhance therapeutic effect. Furthermore, a HDGF sandwich ELISA platform with well linear range between 10-0.15625 ng/ml has been established and able to detect the HDGF level in human and rat serum. Anti-HDGF antibody was also used for extended applications, included in analgesic application and allow to treat cancer stemness and Sorafenib drug resistant Huh7 cells (Huh7-SR). Our finding indicated anti-HDGF hmAb-7 exhibited analgesic effect in rat chronic constrictive injury (CCI) model plus with Adenoviral HDGF gene delivery. Besides, hmAb-7 not only shown sphere and colony inhibitory effects in continuous spheroid cell cultured Huh7 and HepG2 cells, but also inhibited cancerous behaviors in Huh7-SR cells. Finally, adenovirus mediated anti-HDGF-scFv gene delivery suppressed the colony and sphere formation in both Huh7 and Huh7-SR cells. The adenovirus mediated scFv gene delivery provided a novel approach for anti-HDGF therapy and allowed for further basic studies.
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Hasan, Noor Haliza. "Avian Influenza virus M2e protein: Epitope mapping, competitive ELISA and phage displayed scFv for DIVA in H5N1 serosurveillance." Thesis, 2017. http://hdl.handle.net/2440/119643.
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Within the avian influenza virus (AIV) history, H5N1 subtype is the most alarming in terms of its spread rate throughout the globe with its demonstrated unusual pattern of evolution. Persistency and constant circulation of this subtype in poultry population in a number of countries have resulted its establishment and declaration as enzootic. The affected countries are commonly characterised by high poultry populations and productions. They are also developing countries which have minimal funding allocated for precaution on disease incursion. Past observations showed that a single AIV epizootic is capable of causing significant economic burden throughout the world. Although epizootic, it still resulted sporadic cases of human infection and mortality. Therefore, H5N1 enzootic countries opt for vaccination strategy (usually with inactivated whole virus) to evade AIV incursions. However, this interferes with the AIV surveillance effort. This is due to the lack of diagnostic tool with the ability to differentiate AIV infected animal from vaccinated animal (DIVA). Following this realisation, several options are made available. Diagnostic tool development which is capable of DIVA requires a highly sensitive and specific target which at the same time is economic, and pose ease of application. In recent years, growing interest on the AIV matrix 2 extracellular domain (M2e) protein has propelled its exploration as the target for AIV serosurveillance diagnostic tool development. It has been demonstrated to be highly sensitive and specific in detection for AIV infection in an indirect enzyme-linked immunosorbent assay (ELISA) setting. The factor which made it highly interesting is its ability for DIVA application. M2e protein can only be found in low concentration on an AIV particle which is used in an inactivated vaccination strategy, while present in high concentration if cells are AIV infected. Therefore, this study has further explores the AIV M2e protein potential for AIV serosurveillance diagnostic tool development and successfully demonstrated an M2e-based test in a competitive ELISA format for DIVA. This particular ELISA format was of interest as it can be potentially used in multiple species application, as AIV is a multispecies pathogen. To ensure the universality of the competitor antibody, comparative mapping of anti-M2e antibodies from chicken, mouse and rabbit was done. Findings highlighted slight variations in the epitope identified for the M2e antigen by antibodies from different species. Mouse anti-M2e antibodies are more suitable to be used as the competitor antibodies against anti-M2e chicken sera in the M2e-based competitive ELISA test. Consequently, application of the mouse anti-M2e antibodies in the M2e-based competitive ELISA has demonstrated specific and sensitive indication of AIV infection in the H5N1 challenged chicken sera. Biotechnology developments has also introduced the single chain variable fragment (scFv) antibodies as specific and stable bait for antibodies detection against targeted pathogen’s protein (antigen). Taking advantage of this knowledge, this study has also successfully isolated reactive and specific anti-M2e scFv antibodies from avian sources. This is critical as an avian sourced antibodies to be used as bait for the targeted pathogen’s protein is highly relevant in the setting for AIV serosurveillance application in the poultry industry. These findings are significant in the effort to provide a highly sensitive and specific diagnostic tool, which are also cost effective, easy to apply with high throughput ability. Such ideal diagnostic tool for AIV serosurveillance is highly valuable, as this may hold the key to break the AIV continuous circulation.Thesis (Ph.D.) -- University of Adelaide, School of Animal and Veterinary Sciences, 2017
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Lin, Shu-Jian, and 林淑娟. "Construction of scFv Phage Display Library Against Diphenylamine." Thesis, 2004. http://ndltd.ncl.edu.tw/handle/92251249392714967412.
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碩士國立中興大學
生命科學院碩士在職專班
92
Diphenylamine is an antioxidant, having the skeleton structure of non-steroidal anti-inflammatory drugs. In the use of agriculture, it is as a dipping solution to prevent superficial scald in the surface of apple. Diphenylamine also is a fungicide and was used in the pesticides with high concentration. It is an important derivative of BXT, and often used in hair color dye. It is toxic, possible mutagen and teratogen, and harmful in contact with skin. Because diphenylamine is a hapten, in this experiment, we use conjugate reagent to synthesize the bovine serum albumin with diphenylamine together as an immunogen. After immunization, and confirmed with ELISA, we take out the spleen of mouse, and use a series of degenerate primers covering recently known murine immunoglobulin gene family and performing RT-PCR to amplify variable region of heavy chain (VH) and light chain (VL) gene. The amplified VH and VL DNA fragments were assembled to perform single chain fragment of variable region (scFv). The scFv DNA were cloned to the pHEN2 vector, and then transformed to E. coli. We use the technique of phage display system to construct the library of scFv for further selection. The constructed antibody phage display library was confirmed the diversity by BstNI restriction fingerprinting analysis.
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"Improving scFv stability through framework engineering." Thesis, 2012. http://hdl.handle.net/10388/ETD-2012-11-771.
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The availability of cost-effective high throughput screening assays combined with an enhanced understanding of oncogenesis has driven the development of more potent, specific, and less toxic anti-cancer agents. At the forefront of these advances are immunoglobulin molecules and their fragments. However, difficulties in producing antibodies in sufficient quantity and quality for commercial application have driven the development of alternative systems that can produce antibodies efficiently and cost-effectively. This thesis focuses on the engineering of an antibody fragment referred to as a single chain variable fragment (scFv), which consists of antibody light and heavy chain variable domains fused together by a peptide linker.
Although the use of scFvs circumvents many of the issue of full-length antibody production, they still possess their own unique set of difficulties, including stability. In this thesis, we explored the following strategies to increase scFv stability. First, we increased the number of linkers used to join the variable light and heavy domains. We constructed two linear and two cyclic permutated scFvs that contained additional peptide linkers. Two linear permutated scFvs, named Model 1 and Model 3, showed increased stability with calculated melting temperatures (Tms) exceeding that of the unpermutated scFv. The two cyclic scFvs were less stable with Tms less than that of the unpermutated scFv. Second, we mutated light and heavy variable domains by introducing prolines or mutating glycine to alanine in the variable domain framework regions. Sites for proline mutations and glycine to alanine mutations were identified and scFvs containing the mutations were purified and their thermal stability tested. Unfortunately, there were no discernible differences between purified scFv mutants and the control scFv. Third, we designed a new selection/screening strategy using phage display and yeast two-hybrid assays to identify complementarity determining regions on scFvs that increased intracellular stability. We used this strategy to isolate anti-Abl-SH3 scFvs. Transient expression of scFvs in K562 cells indicated that two anti-Abl-SH3 scFv decreased viability.
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Yi-WenChang and 張憶文. "Discovery of antibodies by phage display human scFv antibody library." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/15750785477965469165.
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huang, chi hung, and 黃濟鴻. "Generation of scFv against Thalidomide stereoisomer from phage display library." Thesis, 2003. http://ndltd.ncl.edu.tw/handle/65105760400880058834.
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碩士國立中興大學
生物化學研究所
91
Thalidomide was first introduced in the 1950s as a sedative drug. Because it was deemed to be safe, it was prescribed for nausea and insomnia in pregnant women. However, it was found to cause the severe birth defects in children whose mothers had taken the drug in their first trimester of pregnancy. This was a terrible tragedy. Deepgoing research realized that one of thalidomide enantiomer possesses the desired therapeutic effect of quelling nausea, but its mirror compound produces fetal deformities. In this work, we synthesis thalidomide conjugated with BSA as an immunogen in order to produce related antibodies. After immunization, RT-PCR was performed and a series of primers was used to amplify splenic VH/VL genes. Then, amplified VH/VL were assembled to construct the antibody phage display library against thalidomide. The constructed antibody phage display library was being selected against immunogen after comfirming the diversity by BstNI restriction enzyme fingerprinting analysis. After three rounds of selection, we have isolated two phage clones with weak affinity against (+) or (-) thalidomide individually. Colony PCR and western blotting analysis showed these two clones bear full-length of scFv DNA fragment and are able to express scFv. Competitive ELISA assay also confirmed the specificity of these two scFvs against (+)-thalidomide or (-)-thalidomide respectively.
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Ying-HsiuLiu and 劉盈秀. "Selection and characterization of EV71 antibody by phage display human scFv antibody library." Thesis, 2013. http://ndltd.ncl.edu.tw/handle/42895278044035675388.
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碩士國立成功大學
醫學檢驗生物技術學系碩博士班
101
Enterovirus 71 (EV71) which causes hand-foot-mouth disease (HFMD) and herpangina belongs to the Picornaviridae family with ss(+) RNA virus. In 1998, EV71 outbreak in Taiwan and resulted in 78 fatal cases of 405 severe complications related neurological or cardiopulmonary symptoms. EV71 has been threatened the children under 3 years of age in Asia-Pacific area. Vaccination of EV71 seems the most efficient to eradicate the infectious disease. However, the development of vaccines is still in clinical trials. Antibodies also develop for early detection and diagnosis to control and prevent EV71 transmission. Different antibody format can be expressed by phage display technology via genetic engineering. Here, we selected the phage clones to against EV71 from the human scFv antibody library via biopanning. Procedure of biopanning included binding the target, washing out unbound phage and amplification of binders. Phage clones in total 385 were random picked and grouped by RFLP with SalI restriction enzyme. Five groups were identified and sequenced the nucleotides. Sequence data were aligned with the international ImMunoGeneTics information system (IMGT). We found that all of five phage clones contain a variable heavy sequence. Phage clone 3 and 9 contained full length scFv nucleotide sequence but formed no functional scFv antibody in protein level. However, the phage clones 6 and 13 showed binding ability to against EV71 that revealed in ELISA results. In addition, the phage clone 6 and 13 can recognize EV71virus particles and VP2 protein in western blot. Phage clone 6 and 13 detected EV71 in RD cell can be observed by immunofluorescence assay (IFA). In this study, the data suggested that phage clone 6 and 13 from the human scFv antibody library can be applied for EV71 research or diagnosis.
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Chiang, Wei-chung, and 姜為中. "Construction and Selection of Antibody Against N-Acyl-L-homoserine lactone from scFv Phage Display Library." Thesis, 2003. http://ndltd.ncl.edu.tw/handle/91891674175611937800.
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碩士國立中興大學
生物化學研究所
91
Many kinds of microorganisms are capable of regulating genes by Quorum sensing (QS) mechanism. When the cell density reaches at a certain level high, the quorum sensing regulated genes will be activated. In fact, quorum sensing genes are trigerred by organic signal molecules that are able to freely diffuse though the cell membrane. The signal molecules, also called autoinducer (AI), are derivatives of acyl homoserine lactone (AHL). Virulence genes found in many pathogenic bacteria were recently proved to be associated with AHL regulated QS system. Since AHLs regulate most virulence activity of pathogenic mechanisms, it might be significant to neutralize AHL in the course of prevention bacteria infection. Since AIs have common structure, homoserine lactone ring, we have designed and synthesized homoserine lactone ring conjugated with succinyl-BSA as an immunogen in order to produce antibodies which can bind to variety of AHL. After immunization, we use a series of degenerate primers covering recently known murine Ig gene family and performing RT-PCR to amplify splenic VH/ VL genes. Then, the amplified VH/ VL were assembled to construct an antibody phage display library. In the preliminary work we found the complexity and variation of germline VH/ VL DNA increase the difficulty of DNA manipulation, sometimes even hinder the reaction. After repetitive attempts, we have gained the best reaction parameters. The constructed antibody phage display library was being selected against immunogen after comfirming the diversity by BstNI restriction fingerprinting analysis. After three rounds of selection, we have isolated three phage clones with weak affinity against AHL. Further analysis showed these clones bear full-length of scFv DNA fragment and are able to express scFv. After transforming to the other E. coli suitable of protein expression, we will assess their ability of inhibiting QS system and evaluate the potential of medical use.
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Marques, Gonçalo Rodrigues Puidival. "Development of antibodies against the Notch pathway ligand JAG1 using phage display technology." Master's thesis, 2019. http://hdl.handle.net/10362/94394.
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The Notch signaling pathway is a cell-to-cell communication system that plays crucial roles during the embryonic development and in the tissue homeostasis. In mammals, this pathway is constituted by four Notch receptors (Notch1-4) and five Notch ligands (DLL1, 3 and 4, and JAG1 and 2). Binding of the ligands to the receptors in adjacent cells leads to the activation of the Notch pathway and the regulation of a multitude of genes that control many cellular processes such as stem cell self-renewal, cell differentiation, proliferation, and survival. Deregulated expression of Notch signaling components are observed in many cancers, including breast, and shown to be implicated in tumor growth, recurrence and drug resistance. JAG1 is one of the five Notch ligands that is overexpressed in aggressive cancers and mediates many of the Notch signaling tumorigenic functions. JAG1 overexpression promotes cancer cell survival, proliferation, migration, metastasis, cancer stem cell population maintenance, tumor-associated angiogenesis, and allows tumor cells to escape the immune surveillance.
The goal of this work was to develop specific anti-JAG1 antibodies using phage display technology. To achieve that we used the Human Single Fold scFv Tomlinson library I+J and recombinant JAG1-EGF3-Fc protein as antigen. After several rounds of selections, 84 scFv clones capable to recognize and bind to recombinant JAG1 proteins were isolated. The binding of the 84 scFvs towards the JAG1 was tested by ELISA assays using recombinant JAG1 proteins. Additionally, the binding of anti-JAG1 scFvs to endogenous cellular JAG1 was tested by flow cytometry. Sequencing analysis of the selected clones allowed the identification of 19 unique anti-JAG1 scFvs. One was reformatted into two anti-JAG1 IgGs: one with a glycosite in HCDR2 and the other without. These IgG molecules were characterized by ELISA and SDS-PAGE, and our data showed that after reformatting they were no longer able to recognize JAG1.
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Gonçalves, Vasco Miguel Reis. "Targeting membrane proteins of latently infected CD4+T cells providing a starting point to the eradication of HIV-1." Master's thesis, 2013. http://hdl.handle.net/10451/15861.
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Tese de mestrado, Ciências Biofarmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2013AIDS is characterized by an immunodeficiency developed due to an infectious disease caused by a world spread virus, the HIV‐1. Infection with this virus culminates invariably in the death of the infected individual due to opportunistic infections that find a way into the organism. Today, HIV‐1 is fought against with therapies based in antiretroviral drugs that, although efficient and able to stop or difficult the viral infectious cycle, do not provide a real cure for AIDS, this is, HIV‐1 infection is still considered a chronic and fatal condition. The incapacity of such therapies to eradicate HIV‐1 infection is in part due to the existence of viral reservoirs like the CD4+ T cells population which are responsible for maintaining the HIV‐1 in a latent state inside the cell, protecting it from the host immune system and administered drugs, waiting for some trigger capable to reactivate it. This project was born from the possibility to achieve a sterilizing cure upon finding that specific trigger, enabling the depletion of such reservoirs and subjugating the reactivated HIV‐1 to the effects of the antiretroviral drugs. For this, we traced and objective in selecting antibodies through a run of the mill technology, the phage display technique, against unknown membrane determinants of the latently infected cells searching for agonist antibodies capable of reactivating the latent HIV‐1 and possibly, bringing us one step closer to a cure for AIDS. scFv of human origin were selected against J‐Lat 10.6 and their agonist capacity in causing a phenotypic modification was evaluated resorting to flow cytometry. The work here presented permitted us to identify four promising scFv with agonist HIV‐1 latent reactivation capacities against J‐Lat 10.6 cells and, additionally demonstrated the possibility for our simple approach to be an alternative methodology to the lentiviral agonist selection method developed by Hongkai Zhang and associates.
A SIDA, ou síndrome da imunodeficiência humana adquirida desenvolve‐se devido a uma doença infeciosa de incidência mundial causada pelo vírus da imunodeficiência humana, o VIH tipo‐1. Este vírus tem um tropismo específico para células do sistema imunitário, o que leva à depleção desta população celular inevitavelmente debilitando as defesas naturais do organismo. Tal estado imunodeprimido contribui para que infeções oportunistas se instalem, causando complicações adicionais o que resulta invariavelmente na morte do individuo seropositivo. Até à data os doentes com VIH‐1 são sujeitos a terapias que se baseiam em combinações de fármacos antirretrovirais e que, apesar de eficientes bloqueando ou dificultando o ciclo infecioso do vírus em algum momento, não constituem uma verdadeira cura para a SIDA. A infeção com VIH‐1 continua a ser considerada uma condição crónica e fatal. A dificuldade que tais terapias têm em erradicar a infeção causada pelo VIH‐1 é em parte devida à existência de reservatórios virais, como a população de células T CD4+. Esta células inadvertidamente mantêm o VIH‐1 num estado latente no interior da célula, protegido do sistema imunitário do hospedeiro e de qualquer terapêutica farmacológica, aguardando por algum evento desencadeador do processo de reativação. É derivado desta observação fisiológica que este projeto nasce. A partir da possibilidade de se obter uma cura esterilizante ao se encontrar um gatilho capaz de reactivar o VIH‐1 latente e que permita então a depleção dos reservatórios de latência viral e submeta o HIV‐1 à mercê dos fármacos antirretrovirais. Para tal traçamos um objetivo baseado numa técnica laboratorial corriqueira, o phage display, para se selecionar anticorpos contra determinantes membranares apresentados pelas células latentemente infetadas. A identificação de anticorpos agonistas capazes de reativar o HIV‐1 latente num modelo celular laboratorial, as J‐Lat 10.6, aproxima‐nos da possibilidade de encontrar uma cura para a SIDA. Tal identificação foi facilitada recorrendo à técnica de citometria de fluxo que permitiu avaliar a capacidade dos anticorpos selecionados desencadearem a alteração fenotípica desejada. O trabalho aqui apresentado permitiu‐nos identificar quatro scFv agonistas com capacidade de reativar o HIV‐1 latente em células J‐Lat 10.6 e, adicionalmente, demonstrou que pode ser utilizado como uma alternativa à metodologia utilizada por Hongkai Zhang na sua seleção de anticorpos agonistas através do uso de partículas lentivirais.
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Zhou, Jie [Verfasser]. "Generation of scFv recombinant antibodies with the help of the phage display system against the microtubule associated protein Tau and the kinase MARK / presented by Jie Zhou." 2000. http://d-nb.info/960264000/34.
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"Antibody Based Diagnostic and Therapeutic Approach for Alzheimer's Disease." Doctoral diss., 2014. http://hdl.handle.net/2286/R.I.27402.
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abstract: Alzheimer's disease (AD) is the most common form of dementia leading to cognitive dysfunction and memory loss as well as emotional and behavioral disorders. It is the 6th leading cause of death in United States, and the only one among top 10 death causes that cannot be prevented, cured or slowed. An estimated 5.4 million Americans live with AD, and this number is expected to triple by year 2050 as the baby boomers age. The cost of care for AD in the US is about $200 billion each year. Unfortunately, in addition to the lack of an effective treatment or AD, there is also a lack of an effective diagnosis, particularly an early diagnosis which would enable treatment to begin before significant neuronal damage has occurred.
Increasing evidence implicates soluble oligomeric forms of beta-amyloid and tau in the onset and progression of AD. While many studies have focused on beta-amyloid, soluble oligomeric tau species may also play an important role in AD pathogenesis. Antibodies that selectively identify and target specific oligomeric tau variants would be valuable tools for both diagnostic and therapeutic applications and also to study the etiology of AD and other neurodegenerative diseases.
Recombinant human tau (rhTau) in monomeric, dimeric, trimeric and fibrillar forms were synthesized and purified to perform LDH assay on human neuroblastoma cells, so that trimeric but not monomeric or dimeric rhTau was identified as extracellularly neurotoxic to neuronal cells. A novel biopanning protocol was designed based on phage display technique and atomic force microscopy (AFM), and used to isolate single chain antibody variable domain fragments (scFvs) that selectively recognize the toxic tau oligomers. These scFvs selectively bind tau variants in brain tissue of human AD patients and AD-related tau transgenic rodent models and have potential value as early diagnostic biomarkers for AD and as potential therapeutics to selectively target toxic tau aggregates.Dissertation/Thesis
Doctoral Dissertation Chemical Engineering 2014