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Dissertations / Theses on the topic 'Antibody-Libraries'

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Published: 1 April 2023

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1

Charlton, Keith Alan. "Combinatorial antibody display libraries from sheep and their analysis." Thesis, University of Aberdeen, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.342711.

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2

Büssow, Konrad. "Arrayed cDNA libraries for antibody screening and systematic analysis of gene products." [S.l. : s.n.], 1998. http://www.diss.fu-berlin.de/1999/53/index.html.

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3

Skamaki, Kalliopi. "In vitro evolution of antibody affinity using libraries with insertions and deletions." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/286439.

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In Nature, antibodies are capable of recognizing a huge variety of different molecular structures on the surface of antigens. The primary factor that defines the structural diversity of the antibody antigen combining site is the length variation of the complementarity determining region (CDR) loops. Following antigen stimulation, further diversification through the process called somatic hypermutation (SHM) leads to antibodies with improved affinity and specificity. Sequence diversification by SHM is mainly achieved by introduction of point substitutions and a small percentage of insertions/deletions (indels). Although the percentage of indels in affinity matured antibodies is low, probably due to the low rate incorporation of in-frame indels throughout the course of the SHM diversification process, it is likely that the antibody fold can accommodate higher diversity of affinity-enhancing indels. By in vitro evolution, other researchers have sampled either only restricted diversity of indels or extended diversity of insertions only in specific positions chosen based on structural information and natural length variation. The aim of this thesis was to study the impact of random and high diversity indels on antibody affinity by in vitro evolution. New approaches for construction of libraries with in-frame amino acid indels were applied to enable sampling of indels of different lengths across the entire antibody variable domains. I followed two different approaches for construction of indel libraries. Firstly, a recently developed random approach allowed the construction of libraries with random insertions and deletions. Secondly, a semi-random approach was developed to build libraries with different lengths of insertions that could be widely applied in future in vitro antibody affinity maturation campaigns. Libraries constructed by either of these approaches yielded variants with insertions with improved affinity. Overall, this thesis demonstrates that insertions besides offering alternative routes to affinity maturation can also be combined with point substitutions to take advantage of additive effects on function.
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4

Sblattero, Daniele. "The Development of a Single Vector Recombination System to Make Large Phage Antibody Libraries." Doctoral thesis, SISSA, 1999. http://hdl.handle.net/20.500.11767/4385.

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1.1.1 The aim of the reasearch Phage display has been recently introduced as a means of making antibodies in vitro(Barbas, Kang et al. 1991; Griffiths, Malmqvist et al. 1993; Griffiths, Williams et al. 1994; Vaughan, Williams et al. 1996; Sheets, Amersdorfer et al. 1998). In general, the affinity of the antibodies isolated is pfoportional to the initial size of the library used for selection. This is based on both theoretical (Perelson and Oster 1979) and practical (Vaughan, Williams et al. 1996) considerations. In view of this, the creation of larger and larger libraries has bec01ne an important goal in the use of this technology in the selection of antibodies against any antigen. The aim of my research were: - the design and the validation of a new phagemid vector for phage display of antibody fragments (scFvs). - The design of a new strategy for "one vector" in viva recombination. - The construction of a large naive scFv libray 1.1.2 Results obtained The following chapters will describe all the steps taken in order to achieve the final goal: the construction of a large phage antibody library. First of all there will be the description of the design of a new phagemid vector in which all the possible variables were considered from a theoretical point of view, and the best elements were chosen for use. Secondly data confirming the quality of the vector will be pre_sent.ed. __ This was achieved first with the cloning of several mAb and then with the construction of two small immune libraries that allow the selection of several specific and functional scFv. The third part will describe a method which uses a single vector to exploit the reversibility of ere catalysed recombination. This method involves the creation of a relatively small primary library (7xl o7 was used here) in a phagemid vector in which the VH and VL genes are separated by two nonhomologous lox sites. The heavy and light chain genes in this primary library are then recombined by infecting the phagemid particles into ere expressing bacteria at high multiplicity of infection (MOI). Under these conditions many different phagemid particles enter a single bacteria and the VH and VL genes are exchanged between different phagemids, creating many new VH/VL combinations, all of which are functional. This ends with the production of a large naive library of preaviously unattainable size that was validated by the selection of antibodies, with high affinity, against a large number of different protein antigens. In order to fully appreciate the different aspects of experimental works and its underlying strategies some theoretical paragraphs are included in this first chapter.
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5

Prendergast, D. "Discovery of tumour necrosis factor receptor-1 (p55) binding peptides using a phage display library." Thesis, Queen's University Belfast, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.368468.

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6

Persic, Lidija. "The selection of specific phage antibodies from phage antibody libraries and their expression in different systems." Thesis, Open University, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.299004.

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7

Chiliza, Thamsanqa Emmanuel. "Antibody phage-displayed libraries derived from chicken immunoglobulin genes a source of highly specific diagnostic antibodies /." Diss., Pretoria : [s.n.], 2007. http://upetd.up.ac.za/thesis/available/etd-07012008-080736/.

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8

Alturki, Norah. "Expression of Biotinylated Multivalent Peptide Antigens in Bacteria for Rapid and Effective Generation of Single Domain Antibodies from Phage-displayed Antibody Libraries." Thesis, Université d'Ottawa / University of Ottawa, 2012. http://hdl.handle.net/10393/23523.

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In the present study, two insulin-like growth factor-binding protein 7 (IGFBP7) C-terminal-peptides were expressed as fusion proteins to bacterial verotoxin pentamerization domain as shown by Western blotting, ELISA and mass spectroscopy. Both in vivo-biotinylated recombinant products were purified from bacterial lysates by IMAC and used directly for panning along with the recombinant IGFBP7 protein using the LAC-M Camelidae naïve single domain antibody (sdAb) library. Target-specific sdAbs to both parental protein and peptide fusions were identified by phage ELISA. Twelve different clones were isolated by phage-ELISA screening and their sdAb genes were sequenced. Soluble sdAbs and their pentameric formats were expressed in TG1 E. coli, purified by IMAC and characterized by ELISA and SPR. Several sdAbs are currently under study, however anti-IGFBP7 (P12/M12) was extensively characterized and exhibited promising anti-tumorigenic effect on PANC-1 cell lines by blocking IGFBP7 promoting activity. This study provides the basis for developing a novel imaging/therapeutic reagent for targeting and treating brain tumor angiogenesis in early stages of tumorogenesis and can also be used as a molecular tool to monitor the degree of angiogenesis in gliomas which may help to improve the clinical management of brain tumors.
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9

Diebolder, Philipp [Verfasser], and Roland E. [Akademischer Betreuer] Kontermann. "Generation of "LYmph Node Derived Antibody Libraries" (LYNDAL) : a concept for recovering human monoclonal antibodies with therapeutic potential / Philipp Diebolder ; Betreuer: Roland E. Kontermann." Stuttgart : Universitätsbibliothek der Universität Stuttgart, 2014. http://d-nb.info/112545069X/34.

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10

Giudice, Anna Maria <1991&gt. "ABC transporters of the A subfamily: potential prognostic markers and candidates for antibody selection from phage-display libraries for therapeutic use in Ewing sarcoma." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2020. http://amsdottorato.unibo.it/9421/1/Giudice_AnnaMaria_tesi.pdf.

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The prognostic value of ABC transporters in Ewing sarcoma is still poorly explored and controversial. We described for the first time the impact of various ABCs on Ewing sarcoma prognosis by assessment of their gene expression in two independent cohorts of patients. Unexpected associations with favourable outcomes were observed for two ABCs of the A-subfamily, ABCA6 and ABCA7, whereas no associations with the canonical multidrug ABC transporters were identified. The ABCs of the A-subfamily are involved in cholesterol/phospholipids transportation and efflux from cells. Our clinical data support the drug-efflux independent contribution to cancer progression of the ABCAs, which has been confirmed in PDX-derived cell lines. The impact of these ABCA transporters on tumor progression seems to be mediated by lowering intracellular cholesterol, supporting the role of these proteins in lipid transport. In addition, the gene expression of ABCA6 and ABCA7 is regulated by transcription factors which control lipid metabolism: ABCA6 was induced by the binding of FoxO1/FoxO3a to its promoter and repressed by IGF1R/Akt signaling, whereas the expression of ABCA7 was regulated by p53. The data point to ABCA6 and ABCA7 as potential prognostic markers in Ewing sarcoma and suggest the IGF1/ABCA/lipid axis as an intriguing therapeutic target. Agonist monoclonal antibodies towards ABCA6/7 or inhibitors of cholesterol biosynthesis, such as statins or aminobiphoshonates, may be investigated as therapeutic options in combination with chemotherapy. Considering that no monoclonal antibodies selectively targeting extracellular domains of ABCA6/7 are available, the second part of the project has been dedicated to the generation of human antibody phage-display libraries as tools for selecting monoclonal antibodies. A novel synthetic human antibody phage-display library has been designed, cloned and characterized. The library takes advantages of the high variability of a designed naïve repertoire to be a useful tool for isolating antibodies towards all potential antigens, including the ABCAs.
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11

Zilkens, Kilian Johannes Carl Verfasser], Stefan [Akademischer Betreuer] [Dübel, and Andreas [Akademischer Betreuer] Gerstner. "Identification of novel tumor associated proteins in head and neck cancer using patient derived fresh tissue cDNA libraries and antibody repertoires / Kilian Johannes Carl Zilkens ; Stefan Dübel, Andreas Gerstner." Braunschweig : Technische Universität Braunschweig, 2020. http://d-nb.info/1217402411/34.

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12

Daugherty, Patrick Sean. "Screening combinatorial polypeptide libraries using bacterial surface display and fluorescence-activated cell sorting /." Digital version accessible at:, 1999. http://wwwlib.umi.com/cr/utexas/main.

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13

Rete, Cristian-Victor. "Libraries of dynamic peptides based on reversible native chemical ligation." Thesis, Strasbourg, 2018. http://www.theses.fr/2018STRAF032.

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La possibilité d'utiliser une nouvelle méthodologie pour l'échange de liaisons peptidiques dans des conditions aérobiques et biocompatibles a été étudiée. Nous décrivons l'optimisation de la ligation chimique native combinatoire dynamique (dynNCL) au niveau d'un résidu N-méthylcystéine en utilisant des peptides modèles. Nous employons en outre cette méthode optimisée pour le criblage de bibliothèques de peptides combinatoires dynamiques en présence et en l'absence d'un gabarit de type anticorps. L'effet que l'incorporation d'une jonction dynamique au sein de ligands non-anticorps a sur l'affinité a également été étudié. Nous proposons que dynNCL peut être utilisé pour la conception d'une nouvelle classe de séquences pouvant potentiellement conduire au premier exemple d'épissage de protéines artificielles
The possibility to use a new methodology for peptide bond exchange in aerobic biocompatible conditions has been investigated. We describe the assay optimization of dynamic combinatorial native chemical ligation (dynNCL) at the N-methyl-cysteine residue using model peptides. We further employ this optimized method for the screening of dynamic combinatorial peptide libraries in the presence and the absence of an antibody template. The effect that dynamic junction incorporation into non-antibody ligands has upon affinity was also studied. We propose that dynNCL can be used for the creation of a new class of designer sequences which can potentially provide the first example of artificial protein splicing
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14

Cobaugh, Christian Wessel 1971. "Single scaffold antibody libraries created with high rates of mutagenesis or diversity focused for peptide recognition." 2007. http://hdl.handle.net/2152/15990.

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This dissertation describes several strategies used to create diversity in non-immune antibody libraries. Two of the strategies were used to create two separate peptide focused libraries. Both of these strategies used to create these antigen-class focused libraries used a single scaffold antibody gene that contained diversity only in the variable heavy region. The scaffold antibody gene one of the libraries, the M:anti-pep library, was chosen based on hypervariable loop canonical structures that are characteristic of other anti-peptide antibodies. Additionally, all of the contact residues of this antibody are commonly used contact residues in other anti-peptide antibodies. These positions and others were varied to incorporate the natural diversity of other anti-peptide antibodies. The second library, the Hu:anti-pep, is based on a widely used, unique combination of human germline antibody segments that express well in bacterial expression. Positions were chosen for variation based on their usage as contact residues in both anti-peptide and anti-protein antibodies. The diversity was less focused than with the M:anti-pep library, incorporating all 20 amino acids at "high usage" positions and only four amino acids at "low usage" positions. Both libraries were validated by phage display selections against the peptide angiotensin (AT) and neuropeptide Y (NPY). The M:anti-pep library yielded specific antibodies to both peptides with dissociation constants as low as 14 nM against AT and 18 nM against NPY. The Hu:anti-pep library yielded specific clones with higher dissociation constants: 49 nM against NPY and 11 [mu]M against AT. The final strategy used to introduce diversity is widely used for affinity maturation of low affinity, previously selected antibodies. Extremely high rates of mutagenesis (2.2% of the gene to 2.7%) were used to create two libraries of the anti-digoxin antibody 26-10. The libraries had been screened by others in an attempt to examine the effects of highrates of mutagenesis on the directed evolution of an antibody. A total of 91 isolated clones from both libraries were sequenced. Several consensus mutations were identified near the CDRH3 in the isolated clones, indicating that they had a positive, selectable effect. This study confirmed that high-error rate antibody libraries contain more active clones than expected. Combinations of the selected consensus mutations from these libraries provide moderate enhancements to the kinetics and expression of the wild-type antibody in a non-synergistic manner.
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15

Büssow, Konrad [Verfasser]. "Arrayed cDNA libraries for antibody screening and systematic analysis of gene products / Konrad Büssow." 1998. http://d-nb.info/960757937/34.

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16

Shih, Yi-Ning, and 施憶寧. "The selection and characterization of Fab fragments for hepatitis C virus NS5A from phage display antibody libraries." Thesis, 2002. http://ndltd.ncl.edu.tw/handle/19304418441007595951.

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碩士
台北醫學院
細胞及分子生物研究所
90
Hepatitis C virus (HCV) is the major source of non-A, non-B hepatitis, which causes not only acute hepatitis by transient infection, but also chronic hepatitis, liver cirrhosis and hepatocellular carcinoma in human. Nonstructural protein 5 (NS5) of HCV possesses RNA polymerase activity and plays a key role in viral replication. In this study, we utilized two phage display antibody libraries to generate and analyze antibodies specific for NS5A. The sizes of antibody libraries containing kappa and lambda light chain are 1.3×107 and 2.1×106 pfu (plaque forming unit), respectively. Sequence analysis of 20 randomly selected clones indicated that these Fab fragments consisted of four groups, represented by NS5L6, NS5L13, NS5L14, and NS5K8. Of which, six NS5L6 clones contain identical heavy and light chain genes. Moreover, the light chain gene used by NS5L13 is almost identical to that of NS5L6 clones, suggesting that this light chain might be crucial for the NS5A-binding activity. The Fab expression of these chosen clones was verified as a 50 kDa protein on western blot using anti-human k or l light chain antibodies. ELISA results revealed that NS5L6, 13, and 14 all have NS5A-binding specificity comparable to that of sera from HCV-infected subjects. Viewed as a whole, our results suggested that the phage display antibody technology might provide an alternative way for the generation of human specific monoclonal antibodies. These generated antibodies could be useful for the development of therapeutic agents against infectious diseases in the future.
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17

Penner, Gail. "Screening of 2 expression libraries with an antibody to a calcium binding ATPase inhibitor protein and sequencing of the isolated clones." 1989. http://hdl.handle.net/1993/16965.

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18

Huang, Yi-Jen, and 黃怡仁. "Engineering Anti-vascular Endothelial Growth Factor Single Chain Disulfide-stabilized Antibody Variable Fragments (sc-dsFv) with phage-displayed sc-dsFv Libraries." Thesis, 2010. http://ndltd.ncl.edu.tw/handle/37609987889165955111.

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博士
國立陽明大學
微生物及免疫學研究所
98
Abstract: Phage-displayed fusion antibody on virus coat protein is a well established system which can efficiently engineer the antibody from genotype to phenotype to test the antibody physical properties on targeting to the antigen in vitro. The engineered antibody can be applicable in cancer therapy for the reason that antibody can carry effectors as cargos and aim to the specific cancer region in vivo. But in the reports from America Food and Drug Administration (FDA) approved antibody therapy in clinical trail, unknown antibody targeting will cause many unrelated, unexplained symptoms during antibody treatment. In order to reduce the side effects during antibody drug therapy, knowing the mechanism of protein-protein interaction by systematic analysis from experimental results in vitro are urgently needed to shorten time-consuming drug exploitation. Phage-displayed antibody targeting can be mimicked in vitro by panning amino acids library on antibody to a specific interested target. The recognition site on both antibody and antigen can also be analyzed the physicals between amino acid side-chain interactions in protein-protein interface from x-ray co-crystal structure. Thus, it can narrow down the design region for constructing the antibody library. In this study, antibody is modified from Bevacizumab (AvastinTM), which is one of the humanized anti-VEGF (vascular endothelial growth factor) monoclonal antibodies in the IgG form, and is the first approved by the FDA as a first-line treatment for metastatic colorectal cancer in combination with chemotherapy. This antibody-antigen interaction will be used as a model system to solve the problem on the antibody instability in the form of single chain fragment variable domains (scFv) which is shortcut and linked two domains from IgG and is also benefit with penetration in vivo during therapy. The purpose of this research is focus on building the a new phage display platform to enhance the antibody library selection with inter-domain disulfide bond between antibody variable domains to stabilize the scFv, to become the single chain disulfide-stabilized antibody variable fragments (sc-dsFv). In conclusion, the carboxyl terminal of the signal sequence manipulate the mass production of the sc-dsFv expressed on phage reveals a new fusion antibody platform on phage display system for further engineering designs, which is as convenient as the scFv-phage expression system but without the defect in the instability of the scFv. After the selection from phage-displayed sc-dsFv antibody libraries, the monoclonal antibody sc-dsFv can be further mass production with stability.
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19

Pires, Tiago Maria Santos de Ochôa e. Azevedo. "Exploring Japonese quail immune repertoires for antibody discovery." Master's thesis, 2018. http://hdl.handle.net/10316/84753.

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Dissertação de Mestrado em Biotecnologia Farmacêutica apresentada à Faculdade de Farmácia
Avian hosts are widely used for generation of antibodies and enable scalable and cost-effective production in eggs. Notably, hens also enable accelerating monoclonal antibody (mAb) discovery in biopharmaceutical industry since: 1) their phylogenetic distance from mammals ensures generation of robust and specific antibodies against conserved mammalian proteins and 2) the preparation of combinatorial phage-display libraries from chicken sources is simplified over mammalian systems. The present work has explored the potential of Japanese quail (Coturnix japonica) immune repertoires for mAb development. Multiple sequence search and analysis tools were used to revisit C. japonica genome (available since 2013) and identify/characterize genetic regions encoding for the light (LC) and heavy chain (HC) of quail immunoglobulin (Ig), namely using chicken data as reference.We were able to identify two main regions, corresponding to genes encoding quail Ig LC and HC. Simultaneously and supporting the genetic analysis, protein structural homology model predictions also allowed to identify folding conservation within certain domains, when comparing quail and chicken Igs. These supported the design of specific oligonucleotides that were subsequently used in PCR amplification of antibody fragments from a spleen cDNA samples, obtained from hyperimmune birds. Finally, preliminary NextGen sequencing (NGS) analysis of the fragments confirmed the expected distribution of conserved and variable regions.The present study is a relevant contribution for the implementation of methodologies that will allow generation of synthetic immunological repertoires from C. japonica hosts and subsequent development of therapeutic monoclonal antibodies.
A utilização de Aves como animal hospedeiro para geração de anticorpos é uma abordagem largamente explorada, que permite uma produção de anticorpos de baixo custo e escalável em ovos de aves poedeiras. Notavelmente, as galinhas também permitem acelerar a descoberta de anticorpos monoclonais (mAb) na indústria biofarmacêutica, uma vez que: 1) a sua distância filogenética em relação a mamíferos assegura a geração de anticorpos robustos e específicos contra proteínas conservadas de mamíferos e 2) a preparação de bibliotecas combinatórias de Phage-Display a partir de repertórios imunológicos de galinhas é simplificada em comparação com sistemas de mamíferos. O presente trabalho focou-se no estudo do potencial de repertórios imunológicos de codorniz japonesa (Coturnix japonica) no desenvolvimento de mAb. Foram utilizadas nomeadamente múltiplas ferramentas de busca e análise de sequências, para revisitar o genoma da C. japonica (disponível desde 2013) e identificar / caracterizar regiões genéticas que codificam a cadeia leve (LC) e pesada (HC) da imunoglobulina (Ig) de codorniz, usando dados de Galinha (Gallus gallus) como referência. Neste trabalho identificamos duas regiões análogas aos genes que codificam a a LC e HC da Ig de codorniz. Simultaneamente, e suportando a análise genética, previsões do modelo de homologia estrutural das proteínas codificadas por estes genes, permitiram identificar a conservação na estrutura dos domínios da Ig, entre a codorniz e a galinha. Estas análises permitiram o desenho de oligonucleótidos que foram subsequentemente utilizados na amplificação por PCR dos fragmentos de anticorpo, a partir de amostras de bibliotecas de cDNA do baço previamente obtidos de aves hiperimunes. Adicionalmente, uma análise preliminar por sequenciação NextGen (NGS) destes fragmentos confirmou a distribuição esperada das regiões conservadas e variáveis características de Ig.Este estudo representa uma contribuição relevante para a implementação de metodologias que permitem a geração de repertórios imunológicos utilizando a C. japonica e o posterior desenvolvimento de anticorpos monoclonais terapêuticos.
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20

Petrul, Heike Maria. "Production of recombinant antibody molecules with specificity for proliferation antigens on human endothelium using phage display libraries for the optimization of the therapy of solid tumours and rheumatoid diseases = Herstellung von rekombinanten Antikörpermolekülen mit Spezifität für Proliferationsantigene auf humanen Endothelzellen basierend auf Phagen-Genbanken zur Optimierung der Therapie von soliden Tumoren und rheumatischen Erkrankungen /." 1999. http://www.gbv.de/dms/bs/toc/310687543.pdf.

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