Tesis sobre el tema "CSF single chain library"
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Cortini, Andrea. "Serum profiling and autoantibodies identification in Multiple Sclerosis using epitope and CSF IgG phage display libraries". Doctoral thesis, Università degli studi di Trieste, 2009. http://hdl.handle.net/10077/3073.
Texto completoMultiple sclerosis (MS) is considered the prototype of inflammatory autoimmune diseases of the central nervous system (CNS). The typical feature of the disease is the plaques of demyelination. The evolution of the plaque lesion in MS implicates an inflammatory phase followed by a recovery of functional myelin; a second step is the chronic progressive disease with axonal loss. The earlier phase of MS may be mediated by an autoimmune reaction. Whereas the role of T cells in MS pathogenesis is well established, the role of B cells and autoantibodies in demyelination and plaque formation is still unresolved. However several evidences suggest a contribute of autoantibodies in MS pathogenesis. B cells and myelin specific autoantibodies are present in the sclerosis plaques, and there is an increased production of immunoglobulin (Ig) in the cerebrospinal fluid (CSF) of more than 90% of MS patients . Typically these Ig present an oligoclonal pattern and sequencing of oligoclonal IgG showed extensive somatic mutations suggesting B cell clonal expansion and a specific antigen-driven immune response. The most extensively studied putative autoantigens are components of CNS myelin (myelin basic protein MBP, proteolipid protein PLP, myelin oligodendrocyte glycoprotein MOG). The autoantibodies in MS recognize both linear and conformational epitopes, but at present the conformational epitopes of myelin proteins have not been identified. For example, in MS, the T-cell receptors of autoreactive T lymphocytes recognize various peptides of the MBP, and, in EAE, the anti-MOG antibodies recognize only conformational epitopes. Furthermore, the progression of MS is accompanied by the decline of primary T-cell autoreactivity and by the concurrent emergence of neo-autoreactivity (epitope spreading). However recent investigation have showed that no myelin antigens, like neuron-specific enolase (NSE), retinal arrestin, beta-arrestin, may also have a role in MS pathogenesis. Autoimmunity against these antigens may be linked to neurodegeneration, defective remyelination, and predisposition to uveitis in multiple sclerosis. Several strategies, involving the phage display technology, have been employed in the attempt to discover the antigen that drives the immune response in MS. A first strategy depends on the cloning of IgG repertoire of MS patients in a phage display library screened with brain sections or known antigens. Another strategy involves large phage display libraries of random peptides screened with IgG of CSF in order to identify peptides recognized by antibodies present in CSF of MS patients. Phage display is a technique which involves the coupling of phenotype to genotype in a selectable format. It has been extensively used in molecular biology to study protein-protein interactions and to select antibodies against a wide range of different antigens. In this project we have proposed: 1. to study the autoimmune response in MS by using the phage display for the expression of antibodies involved in the disease. We wanted to make a ScFv library from B cells of CSF of different multiple sclerosis patients, to employ as tool to select a phage display Human Brain cDNA library for the identification of new antigens recognized by the immune sistem in MS patients. 2. To produce single gene mini library of putative antigens (MBP,PLP, MOG) for the generation of epitope chips to use for serotyping the immune response in different patients 3. To investigate the feasibility to use a single gene phage display mini-library as tool for epitope mapping (both linear and conformational) of novels autoantigens 4. To investigate the role of NSE(neuron specific enolase), a new possible no myelin autoantigen in multiple sclerosis, in the pathogenesis of the disease and the usefulness as possible diagnostic marker. Results: Scope 1 B-cells from liquor of two MS patients were centrifuged and the total RNA was extracted from the pellets. Total RNA was retrotranscripted and variable region of heavy and light chain of the antibodies were amplified by PCR. Heavy chain and light chain were assorted and assembled before to be cloned in the phagemid vector pDAN 5. A 2x104 independent clones library was obtained and analyzed by PCR and fingerprinting. A diversity of 30,8% for heavy chain and 72,7% for light chain was established. ScFv library was used to select a phage display Human Brain cDNA library. 17 clones with an high reactivity were obtained and after sequencing 6 clones on 17 have shown to be the same antigen(antigen A ); the reactivity on other two antigens obtained with the selection (antigen B and C) of CSF from 18 MS patients and 16 patients with other neurological disease (OND) was tested by ELISA to evaluate diagnostic value of this protein. The results shown that SM response was statistically different from OND response; the ELISA test gave a specificity of 94,12% and a significance of 53,85 %. The reactivity for the antigen B was also evaluated on sera of MS patients and controls. The MS response was statistically different from OND response and shown a specificity of 97,44% and a significance of 58,62 %. Scope 2&3 We have generated three single gene mini libraries of the major antigens in MS (MBP, MOG and PLP); cDNA of each gene was obtained by RT-PCR and after fragmentation cloned in a phagemid vector (pEP1) to obtain a mini-library for each gene. We have obtained a 2x105 for MBP, 2.4x104 for MOG and 1.6x106 for PLP independent clones library. MBP and MOG libraries were characterized by PCR and fingerprinting. Sequencing analysis shown that the entire MBP transcript variant 7 mRNA (664-1177 nt) and MOG isoform alpha 1 mRNA (262-918 nt) were represented in the respective library. To testing the capacity of selecting a single epitope from our libraries, we have performed a selection test with a commercial monoclonal antibody that recognize MBP 82-98 epitope; after three selection panning all selected clones contain the nucleotidic sequence 906- 956 nt (MBP transcript variant 7 mRNA) which encodes the immunogenic epitope recognized by the monoclonal antibody. Scope 4 The reactivity of sera from 31 MS patients and 14 healthy controls was tested by ELISA on NSE ; statistical analysis of the results shown that the two populations were significantly different.
XXI Ciclo
1981
O'Brien, Siobhan Helen. "A single chain antibody bacteriophage display library from a patient with active uveoretinitis". Thesis, University of Aberdeen, 1999. http://digitool.abdn.ac.uk/R?func=search-advanced-go&find_code1=WSN&request1=AAIU123996.
Texto completoBosompem, Amma N. "Isolation of an anti-CD20 single chain variable fragment from a naïve human phage-scFv library". Connect to this title online, 2007. http://etd.lib.clemson.edu/documents/1202410076/.
Texto completoRoberts, Anthony Simon. "The cloning, characterisation and engineering of an IGF-I-BINDING single chain Fv". Thesis, Queensland University of Technology, 2004. https://eprints.qut.edu.au/15914/1/Anthony_Roberts_Thesis.pdf.
Texto completoRoberts, Anthony Simon. "The cloning, characterisation and engineering of an IGF-I-BINDING single chain Fv". Queensland University of Technology, 2004. http://eprints.qut.edu.au/15914/.
Texto completoPilger, Franziska [Verfasser]. "Construction of an equine antibody library in the single-chain-Fragment-variable format (scFv) to express equine immunoglobulins / Franziska Pilger". Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2021. http://d-nb.info/1230824510/34.
Texto completoLi, Weiyi. "Protein Engineering Hydrophobic Core Residues of Computationally Designed Protein G and Single-Chain Rop: Investigating the Relationship between Protein Primary structure and Protein Stability through High-Throughput Approaches". The Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1398956266.
Texto completoNdlovu, 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.
Texto completoShahsavarian, Melody. "Genesis of immune diversity and selection of catalytic antibodies : a new investigation". Thesis, Compiègne, 2015. http://www.theses.fr/2015COMP2215/document.
Texto completoCatalytic 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
"Construction and Characterization of a Single-Chain Variable Fragment Antibody Library against Fusobacterium nucleatum". 2012. http://hdl.handle.net/10222/15184.
Texto completoFarhan Khan placed second in the International Association for Dental Research/Unilever Hatton Competition in the Senior Basic Science Research Category representing Canada, while presenting the research contained in this dissertation. This international competition took place during the 90th General Session & Exhibition of the International Association for Dental Research in Iguaçu Falls, Brazil in June 2012.
Hsieh, Pey-Rou y 謝珮柔. "Screening and Characterization of the Single-Chain Variable Fragment from Phage Library to Against Cyclic Adenosine Monophosphate". Thesis, 2014. http://ndltd.ncl.edu.tw/handle/hdvevv.
Texto completo國立交通大學
應用化學系碩博士班
102
The study is aimed to screen a single chain antibody fragment (scFv) from phage display library for cAMP recognition. The cAMP derivative (containing alkyl amine moiety) was conjugated on BSA and employed for scFv screening. After 6 rounds of panning, 3 clones (designated as 1D, 2E and 5H) with high titers were obtained. Since 2E exhibited higher titer than the other two clones, 2E clone, virtually composed of only the light chain variable region (VL), was mainly used in this study. The corresponding protein, named as cAMP-VL, was overexpressed and purified. The molecular weight was analyzed (14052.8 Da) by electrospray ionization mass spectrometry and confirmed to be consistent with the theoretical value (14054.3 Da). The binding features of cAMP-VL as free protein or on the surface of phage were further analyzed by ELISA or quartz crystal microbalance (QCM). The experiments were conducted using cAMP as probe for interacting with 2E phage and cAMP-VL. The detection limits of 2E phage and cAMP-VL are 200 phage particle/mL and 0.66 µg/mL, repectively. In the case of QCM analysis, cAMP derivative was immobilized on QCM chip, the dissociation constant (Kd) was estimated to be 74,900 phage particle/mL. In order to investigate the binding specificity of cAMP-VL, several nucleotides (adenosine,ATP,ADP and AMP) and nucleosides were used for competitive binding assessment. Results showed that cAMP-VL possesses high specificity to purine moiety. Further study on structure simulation attempted to sketch the binding domain of cAMP. P59, R61, and E81 were found to be the possible candidates for binding. P59R, R61K and E81Q mutants were constructed and purified for binding study. The outcome showed all three mutants moderately damaged the interaction with cAMP indicating these residues are somewhat important for cAMP binding. More studies will be proposed to improve the binding affinity of cAMP-VL toward cAMP based on the structure simulation or the complex of 3D protein structure.
You, Chiou-Ping y 游秋萍. "Generation and affinities with antigen of single chain variable fragment antibody against Odontoglossum ringspot virus from phage display library". Thesis, 2005. http://ndltd.ncl.edu.tw/handle/96527679347915060625.
Texto completo國立嘉義大學
農業生物技術研究所碩士班
93
Odontoglossum ringspot virus (ORSV) is one of the commonest viral pathogens of the cultivated orchids. Infected plants appear unhealthy and may produce low quality flowers. In this experiment a set of ORSV-specific oligonucleotide primers were designed from the region of the coat protein (CP) gene of ORSV. The ORSV CP gene was cloned into the protein expression bacterial plasmid vector of the gutathione S-transferase(GST)fusion protein expression system. The recombinant ORSV CP was injected into mouse to induce immune response of the animal. The cDNAs of VH and VL of ORSV antibody genes were obtained by using reverse transcription polymerase chain reaction from the total the RNAs that were extracted from the spleen cells of immunized mouse. ScFv(single-chain variable fragment)library of ORSV were constructed with gene splicing by overlap extension. Thirty seven scFvs were selected from ORSV-scFv library following three rounds of affinity selection with ORSV CP as an antigen that was expressed in bacteria. Four scFv antibody have specific binding reaction against ORSV CP was selected. Comparing the sensitivity between scFv antibodies and ORSV polyclonal antibody tisted in enzyme linked immunosorbent assay (ELISA) to detect ORSV in leaf extracts of diseased Phalaenopsis plants. Unfortunately, the affinity between scFv antibody and ORSV was weaker than that between polyclonal antibody to the same antigen. The scFv antibodies and polyclonal antibody appeared to have similar sensitivity limit as low as 10 ng/μl. The results highlight the potential of applying the scFv antibodies in the diagnosis of virus diseases.
Wu, Yi-Chen y 吳伊晨. "Generation and affinities with antigen of single chain variable fragment antibodies against Cymbidium mosaic virus from phage display library". Thesis, 2005. http://ndltd.ncl.edu.tw/handle/01271402034029725220.
Texto completo國立嘉義大學
農業生物技術研究所碩士班
93
The cultivated orchids commonly infected Cymbidium mosaic virus (CyMV) that causes the symptoms of leaf necrosis and flower malformation, the prevalent disease may results in enormous loss of orchid growers. For preventing the disease from spreading, the growers should immediately remove the infested plants from greenhouse. For finding the infested plants ELISA is commonly applied to detect the virus due to the immunodetection is simple and economical. In the experiment, the scFv (single chain fragment variable) antibodies against CyMV were developed with recombinant phage antibody system. The primer pair for cloning CyMV coat protein gene was designed based on the gene sequence, the cloned coat protein (cp) gene 672 bp of CyMV was ligated into the expression vector pGEX of the (glutathione S-transferase) expression system, then CyMV cp was purified from the fusion protein of GST-CyMV cp and it was used as antigen to immunize mouse. The total RNAs was extracted from spleen of the immuned mouse and was reversed transcribed into cDNAs, and the VH and VL were cloned from the cDNAs and were assembled with linker into scFv fragments by fill-in reaction. These scFv fragments were ligated into the phagemid vector pCANTAB 5E that transfected competent E. coli cells, so that the phage-displayed antibodies were bio-panned on immobilized antigen from the phage display scFv antibody library. These recombinant phagemids 5-E12, 1-A4, 1-H1, 1-E11, 2-D5, 4-A3 were screened out based on these vectors expressing the scFv antibodies that showed strong affinity with antigen. Both 5-E12 and 2-D5 clones expressed their scFv antibodies that appeared high affinity with CyMV. The sensitivity of immunodetection of scFv antibody 2-D5 was better 5-E12 due to the immunoassay of 100 ng 2-D5 and 5-E12 scFv antibodies bound to 108 particles of CyMV per well were OD405 0.27 and 0.19, respectively. Comparison of the amino sequences both scFv antibody 2-D5 and 5-E12 that appeared very similar, and there are only 3 amino acids different of 250 amino acids between 2-D5 and 5-E12.
Lipes, BD, YH Chen, H. Ma, HF Staats, DJ Kenan y MD Gunn. "An entirely cell-based system to generate single-chain antibodies against cell surface receptors". Thesis, 2008. http://hdl.handle.net/10161/903.
Texto completoDissertation
Yang, Hui-Ming y 楊慧敏. "Selection And Characterization of Human Single-Chain Fv Antibodies Against MUC18 and Epstein-Barr VirusNuclear Antigen-1 (EBNA-1) from a Phage Display Library". Thesis, 2006. http://ndltd.ncl.edu.tw/handle/28361773613674972090.
Texto completo慈濟大學
微免暨分子醫學研究所
94
A large naïve human single-chain Fv (scFv) phage library was used to search for human monoclonal antibodies against tumor-associated antigens by panning with the purified recombinant proteins expressed in E. coli. The cell surface adhesion molecules MUC18, which is overexpressed in several types of human tumors and an Epstein-Barr virus (EBV) nuclear antigen ENBA-1, which is expressed in all EBV-associated tumors, were selected for this study. After five to six rounds of selection-amplification-rescue, 74 of 82 monoclones of MUC18 (90 %) and 14 of 30 (47 %) of EBNA-1 analyzed bound selectively to MUC18 and EBNA-1 in a phage ELISA. The criteria used to assess the specificity of each monoclone was robust binding to the antigen with ELISA reading at A405 > 0.5. In Western blot analyses, the MUC18 scFv fragment antibodies recognized the MUC18 molecules expressed in a variety of tumor cells including HeLa, NPC, melanoma, and breast cancer cell lines. Similarly, EBNA-1 scFv fragment antibodies detected the EBNA-1 molecules only in EBV-positive cells, but the intensity of bands was very weak. The reason for weak detection could be attributed to the low titers of the scFv antibodies, which were obtained after five rounds of selection, in contrast to MUC18 that had gone through six rounds of selection. Diversity analyses of these antigen-selective individual clones by BstNI fingerprinting and nucleotide sequencing revealed a single distinct scFv fragment of MUC18 clones and 7 distinct scFv fragments of EBNA-1 clones. Protein sequences were deduced from the DNA sequences and fragment regions (FR) and complementarity determining regions (CDR) were also identified. The deduced amino acid sequences from the nucleotide sequences of the MUC18 clones analyzed also revealed a single pattern of both light and heavy chains of V-gene. It appears that most of the MUC18 scFv clones isolated use identical or very closely related V-gene sequences. This may reflect that the isolated clones were highly specific and all recognized a single epitope. Despite the variations in BstNI fingerprintings, nucleotide sequences, and deduced amino acid sequences of 11 EBNA-1 binding clones, only the light chain of the V-gene was detected. These results indicated that EBNA-1 binding clones use only VL sequences. The isolation of soluble scFv fragment phages specific for MUC18 was attempted. Even though we could detect by ELISA the soluble forms of scFv antibodies for MUC18, we were unable to detect the MUC18 molecules by Western blot using the soluble scFv antibodies. These results provide a lead for further development of diagnostic assays and selective tumor targeting immunotherapy. Thus, the scFv fragment phage antibodies selected from the phage display library can be used to deliver highly cytotoxic moieties such as radionuclides, toxin, or chemotherapeutic agents to the tumors expressing MUC18 and EBNA-1.