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1
Ng, King Man. "Anti-neurofascin antibodies". Diss., lmu, 2012. http://nbn-resolving.de/urn:nbn:de:bvb:19-150730.
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2
Austin, Eric B. "Human monoclonal antibodies". Thesis, University of Nottingham, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.276187.
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3
Evans, Rachael Yvonne. "The production of anti-idiotopic antibodies to monoclonal anti-RhD antibodies". Thesis, Lancaster University, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.274194.
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4
Kang, Sun-ah. "Apoptotic Cells, Anti-Phospholipid Antibodies, and Anti-Chromatin Antibodies in Autoimmunity". Diss., Temple University Libraries, 2008. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/15651.
Testo completoAbstract (sommario):
Microbiology and ImmunologyPh.D.
Antiphospholipid antibodies (APAs) are detected in various autoimmune diseases, such as antiphospholipid syndrome (APS) and systemic lupus erythematosus. In addition to their binding to negatively charged phospholipids, APAs often cross-react with other molecules. Their potential biological effects are not fully understood. Apoptotic cells are a potential source of auto-antigens during systemic autoimmunity. Inefficient clearance of apoptotic cells results in the development of autoimmune manifestations and intracellular antigens such as nucleosomes become accessible during apoptosis. We examined a panel of monoclonal APAs generated from NZW/BXSB F1, a strain which spontaneously develops autoimmune symptoms reminiscent of APS. These APAs did not bind to live cells, but reacted strongly with different structures within apoptotic cells. Further analysis with various inhibitors indicated that the binding of APAs to apoptotic cells depends on specific caspase activities and on the modification of auto-antigens by reactive oxygen species (ROS). Therefore, apoptotic cells provide a potential source of APA antigens that may not be limited to phospholipids. Our data also indicate that physical accessibility and apoptosis-specific modification of auto-antigens by caspases or ROS are crucial factors for APA-antigen interactions. Various auto-antibodies such as APAs and anti-chromatin antibodies are pathogenic outcome of chronic autoimmune diseases. Their binding to auto-antigens, presumably exposed on apoptotic cells, elicits subsequent amplification of inflammatory responses, thus worsening disease progression. However, the precise immunological functions of auto-antibodies and the mechanism behind are not fully comprehended yet. We investigated immune responses generated by four different auto-immune complexes (auto-ICs) composed of auto-antibodies and apoptotic cells. In the presence of TLR ligation, the presence of auto-antibodies in auto-ICs amplified immune responses generated by apoptotic cells. In most cases, almost all the auto-ICs tested suppressed IL12, TNFa, while increasing IL10 production from macrophages. Further studies with various anti-Fc?R antibodies implied the essential role of various Fc?Rs in elevation of IL10 by auto-ICs. Studies with Mer-/- macrophages indicated that Mer is also crucial in auto-IC mediated augmentation of IL10 production. However, Mer was dispensable for the suppression of IL12. Taken together, auto-antibodies, by forming immune complexes with apoptotic cells, perform strong immunomodulatory functions. Particular importance is in the role of Fc?Rs and Mer in anti-inflammatory responses generated by auto-ICs. Paradoxical, but indispensible contribution of TLR ligation, especially TLR4, in anti-inflammatory responses generated by auto-ICs suggests that auto-antibodies may work as another layer of defense against endogenous danger signals.
Temple University--Theses
5
Chmura, A. J. "Rational engineering of antibodies with irreversible binding : antibodies with infinite affinity /". Connect to Digital dissertations. Restricted to UC campuses. Access is free to UC campus dissertations, 2001. http://uclibs.org/PID/11984.
Testo completoAbstract (sommario):
Thesis (Ph. D.)--University of California, Davis, 2002.Degree granted in Chemistry. Dissertation completed in 2001; degree granted in 2002. Also available via the World Wide Web. (Restricted to UC campuses).
6
Rada-Briega, Cristina. "Somatic hypermutation of antibodies". Thesis, University of Cambridge, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.318450.
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7
Plumpton, Christopher. "Monoclonal antibodies against phytochrome". Thesis, University of Cambridge, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.358677.
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8
Bentall, Andrew John. "Antibodies in kidney transplantation". Thesis, University of Birmingham, 2015. http://etheses.bham.ac.uk//id/eprint/5817/.
Testo completoAbstract (sommario):
The aim of this thesis is to examine the effect of anti-donor antibodies in the clinical management and outcomes of antibody incompatible kidney transplantation. Initial studies were conducted to improve measurement of anti-ABO specific blood group antibodies. The specificity of antibody binding to blood group antigens (BGA) depended upon the assay platform and the nature of the core structure to which the BGA was bound. A standardised haemagglutination assay had excellent reproducibility, which was then applied to the analysis of samples derived from a study of 100 ABO incompatible kidney transplantation (ABOiKTx) in the UK where good clinical outcomes were achieved but there was wide variation reported in local assays quantifying BGA specific antibodies, without survival differences. In a highly sensitised HLA incompatible kidney transplant recipients (HLAiKTx), I demonstrated long term outcomes were poor compared to a compatible cohort, in particular with pre-formed donor specific anti-HLA Class II antibodies, in which histological injury of antibody damage occurred significantly earlier than with Class I antibodies. Further studies demonstrated that anti-HLA antibodies were associated with an inflammatory phenotype, but anti-donor ABO specific antibodies did not despite the activation of complement. Thus, inhibiting terminal complement activation, whilst reducing early antibody-mediated rejection did not abrogate all inflammation which was associated with the presence of IgM DSA. Reproducible and standardised assays are needed for antibody assessment in order to make good clinical decisions to improve patient outcomes. Further studies are needed to stop production or block mechanisms of ongoing cellular infiltrate to improve patient outcomes.
9
Alcocer, Marcos J. C. "Wheat peptides and antibodies". Thesis, University of East Anglia, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.306066.
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10
Farzad, Zohreh (Emami Aleagha). "Studies on anti-tetanus antibodies". Thesis, University of Edinburgh, 1985. http://hdl.handle.net/1842/23886.
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11
Gibb, Alan Patrick. "Cross-reactive antibodies to lipopolysaccharide". Thesis, University of Edinburgh, 1993. http://hdl.handle.net/1842/28093.
Testo completoAbstract (sommario):
Lipopolysaccharide (LPS), also known as endotoxin, is a constituent of the outer membrane of gram-negative bacteria which is toxic for humans and other animals. LPS probably plays a key part in the pathogenesis of Gram-negative bacteraemia and sepsis syndrome in humans. Cross-reactive antibodies to LPS may play a part in natural host defences, and may also be useful in the treatment of Gram-negative bacteraemia and sepsis syndrome. The structure of LPS, its toxicity, its role in Gram-negative bacteraemia and sepsis syndrome in humans, and the potential value of cross-reactive antibodies to LPS are reviewed. The antibody response in recipients of typhoid vaccine was studied, with particular reference to the possibility that typhoid vaccine might induce the production of antibodies to the core region of LPS (LPS-core). In most recipients however the response observed was directed against specific antigens. Urine samples from patients with suspected UTI were tested for IgG antibodies to LPS-core. Such antibodies were found to be associated with the presence of bacteriuria, although the association was not strong enough for antibody assay to be useful as a diagnostic test. Total urinary IgG was equally strongly associated with bacteriuria. This suggested that the antibodies were probably present because of non-specific leakage of serum components into the urine as a result of inflammation. A large number of murine monoclonal antibodies (MAbs) to LPS-core had been produced by a collaborative group in Edinburgh and Basel, in the hope of producing a cross-reactive MAb which would be useful therapeutically. The binding of some of these MAbs to a collection of blood-culture isolates of Gram-negative bacteria was studied.
12
Sheikholvaezin, Ali. "Recombinant antibodies and tumor targeting". Doctoral thesis, Umeå : Univ, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-875.
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13
Benjamin, Richard John. "Tolerance induction with monoclonal antibodies". Thesis, University of Cambridge, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.253988.
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14
Qin, Shi-Xin. "Transplantation tolerance with monoclonal antibodies". Thesis, University of Cambridge, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.305697.
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Rai, Rajendra Singh. "Antiphospholipid antibodies and recurrent miscarriage". Thesis, Imperial College London, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.392475.
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Roberts, S. "Studies on genetically engineered antibodies". Thesis, University of Oxford, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.379906.
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Dalton, Paola. "Maternal antibodies to fetal antigens". Thesis, University of Oxford, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.270344.
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18
Hackett, Gavin S. "Intracellular delivery of therapeutic antibodies". Thesis, University of Nottingham, 2012. http://eprints.nottingham.ac.uk/12611/.
Testo completoAbstract (sommario):
Therapeutic antibodies are highly versatile macromolecules that can be engineered to bind and inhibit a target with high specificity. Unfortunately, the cell membrane is impenetrable to antibody reagents, thus limiting their use almost entirely to extracellular targets. Expanding the application of therapeutic antibodies to intracellular targets is an exciting concept that could have a huge impact on how intracellular protein-protein interactions involved in diseases can be modulated. The modification of therapeutic antibodies with Cell Penetrating Peptides (CPPs) can enable cellular penetration, however, no general approach to modifying antibodies with CPPs has been developed that allows for systematic optimisation of both the in vivo and cell penetrating properties. In this study, neutralising single-chain variable fragment antibodies (scFvs) have been isolated from naïve scFv libraries using antibody phage display that are specific to the model intracellular targets Bcl-2 and Bcl-xL. Lead scFvs showed potent inhibition of these proteins in an in vitro assay with IC50 values of <10 nM being calculated, which is superior to the small molecule Bcl-2/xL inhibitor ABT-737. The lead anti-Bcl-xL scFv was conjugated to the CPPs octa-arginine, HIV Tat49-57 or Antp52-58. These peptides were synthesised to possess either an N-isobutyryl cysteinyl or N-maleimidopropionyl moiety and a C-terminal lysine residue, allowing for site-specific conjugation to an unpaired cysteine residue introduced to the scFv construct and regioselective introduction of 5-carboxyfluorescein to the CPP, respectively. Live-cell confocal microscopy showed that the scFv-octa-arginine conjugate possessed superior cell entry capabilities compared to the scFv-Tat49-57 and scFv-Antp52-58 conjugates. Further studies using a panel of cancer cell lines are required to determine if the anti-Bcl-xL scFv-octa-arginine conjugate can induce apoptosis through inhibition of cellular Bcl-xL. Additonally, a novel approach to controlling the cell penetrating properties of the CPP octa-arginine has been developed. It was demonstrated that carbamate protection of octa-arginine’s guanidine functionality effectively inhibited its cell entry capabilities. Moreover, esterase-labile acyloxymethyl carbonyl (AM) protecting groups were utilised to protect the guanidine functionality of octa-arginine, inhibiting its cell entry capabilities. In a HPLC based assay it was demonstrated that the AM protected octa-arginine was deprotected by pig liver esterase, suggesting that in vivo deprotection could be achieved by serum esterases. The controlled unmasking of octa-arginine is predicted to increase its circulation time and reduce non-specific tissue uptake in vivo, potentially making this CPP more suitable for in vivo applications. The methodologies utilised for the preparation of scFv-CPP conjugates and developed for the controlled unmasking of octa-arginine in this study will allow for optimisation of the cell penetrating and in vivo properties of this promising class of macromolecular therapeutic, thus providing a gateway to unlocking the immense potential of therapeutic intracellular antibodies. .
19
Rix, K. J. B. "Food antibodies in acute psychoses". Thesis, University of Aberdeen, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.593354.
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20
Stevenson, James Dexter. "Chemiluminescence selection of catalytic antibodies". Thesis, University of Nottingham, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.311765.
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21
Lu, Yanling. "Solution conformation of engineered antibodies". Thesis, University of Nottingham, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.442304.
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22
Jones, D. W. "Factor XII and antiphospholipid antibodies". Thesis, University of Kent, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.311229.
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23
Heron, Andrew David. "The stability of monoclonal antibodies". Thesis, University of Glasgow, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.252169.
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24
Marshall, Ann. "Catalytic antibodies for cancer therapy". Thesis, University of Sheffield, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.299626.
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25
Al-Muzairai, Ibrahim Abdulaziz. "Antiidiotypic antibodies in renal transplantation". Thesis, University of Aberdeen, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.280624.
Testo completoAbstract (sommario):
Blood transfusion given before transplantation may improve subsequent allograft survival but the mechanism of action remains unknown. Several groups have postulated that the blood transfusion effect in renal transplantation is mediated through idiotypic- antiidiotypic antibody interactions which result in specific immunosuppression. These antibodies were found to be directed towards both class I & II MHC antigens. Cyclosporin has been shown to be effective in abrogating a primary immune response (if administered concomitantly with blood transfusions) but not an established response. Recent studies have shown that antiidiotypic activity may be enhanced by cyclosporin, but the mechanism remains unknown. The main aims of the studies presented in this thesis were to investigate the idiotypic-antiidiotypic antibodies interaction in renal transplantation. Potentiating activity ('anti-antiidiotypic antibodies:Ab3') was also detected in non-cytotoxic sera from patients who were previously sensitised. Ab3 activity was induced following DST, but all patients who had this activity underwent successful transplantation. Cytotoxic sera were also found to contain Ab3 activity after absorption with platelets. The presence of Ab3 activity prior to transplantation or its subsequent development had no effect on graft survival or rejection episodes. These studies suggest that one mechanism by which blood transfusions improve graft survival is the development of antiidiotypic antibodies. Cyclosporin may enhance Ab2 activity if given during blood transfusion programmes.
26
Isaacs, John Dudley. "Improving serotherapy with monoclonal antibodies". Thesis, University of Cambridge, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.386115.
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27
Ersoy, Oguz 1968. "Amide hydrolysis by catalytic antibodies". Thesis, Massachusetts Institute of Technology, 1996. http://hdl.handle.net/1721.1/38775.
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28
Qian, Jianing. "Affinity chromatography of camelid antibodies". Thesis, University of Cambridge, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.610171.
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29
Lowe, David Philip. "Characterisation of HLA-specific antibodies". Thesis, University of Warwick, 2013. http://wrap.warwick.ac.uk/58070/.
Testo completoAbstract (sommario):
A successful kidney transplant is the best treatment for established renal failure, yet around 300 patients per annum are denied transplants because they have antibodies, most notably directed against donor HLA or ABO in their blood, which have the potential to cause acute and chronic rejection of the transplant. Such antibodies are present in 25% (roughly 1750 of the 7000 on the kidney transplant waiting list) of the patients listed for a deceased donor transplant. Programmes to remove antibody and transplant patients across HLA antibody barriers have been developed, but are limited by a high rate of acute rejection. This thesis explores the factors which may impact upon the pathogenicity of HLA-specific antibodies and also aims to enhance the understanding of the techniques used in the laboratory to define these antibodies. A range of studies were carried out examining factors such as the IgG subclass composition of the anti-HLA response. Assay variations were designed to enable a higher definition of antibody specificity to be achieved, and for the first time in the literature the direct quantification of HLA-specific antibodies in patient sera was performed. In addition, proof of principle design and testing was carried out on a novel prototype therapeutic device for the selective depletion of HLA-specific antibodies directly from patient plasma and sera. The antibodies isolated from this approach were also used in studies to examine the factors which determine serum cytotoxicity.
30
Paudel, Subhash. "Shear thinning in monoclonal antibodies". Thesis, Kansas State University, 2016. http://hdl.handle.net/2097/32833.
Testo completoAbstract (sommario):
Master of ScienceDepartment of Physics
Jeremy D. Schmit
Antibodies are large Y-shaped proteins which are used by immune system to identify and neutralize pathogens. Monoclonal antibody therapy is used to treat different patient conditions. There are problems associated with the manufacturability and deliverability of mAb solutions due to the viscous nature of the protein. The viscosity of antibody solutions increases with the increase in concentration and decreases with applied shear. We want to know why these behaviours are seen and to address this problem we have developed a theory describing the rapid viscosity increase with increasing concentration. We use the polymer theory to explain this behaviour. Here antibodies are treated as polymers. The length of the polymer depend on the aggregation. The reptation time increases approximately as the cubic power of size of aggregate (N³ ). We see the shear thinning behaviour is dependent on the Ab-Ab binding energy and find the relationship between the size of the aggregate and the binding energy. We find aggregate size and morphology using several models for Ab-Ab interaction sites. We use the head to head binding (fAb-fAb binding) model to describe aggregation state in our viscosity theory. The size of the aggregate and hence the reptation time is captured by the binding energy. When the binding energy increases the zero shear viscosity increases and the reptation time decreases. Likewise when the binding energy decreases the zero shear viscosity decreases and the reptation time increases. We have yet to find the correct exponents for the shear thinning behaviour of different mAbs which would be our future work.
31
Råsander, Mattias. "Competitive evaluation method of antibodies". Thesis, Uppsala universitet, Institutionen för biologisk grundutbildning, 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-418992.
Testo completo
32
Dillon, David. "Protective antibodies in normal pregnancy". Thesis, University of Aberdeen, 1989. http://digitool.abdn.ac.uk/R?func=search-advanced-go&find_code1=WSN&request1=AAIU028047.
Testo completoAbstract (sommario):
The aim of this study was to examine the maternal immune response to paternal antigens expressed by the fetus and identify the antigen inducing the response. Sera removed from responder female mice were tested for activity against paternal target cells using a cellular ELISA. Avtivity was first detectable at day 10 of a first pregnancy. The antibody detected in this way was shown to be non-cytotoxic, consisting of the IgGl subclass, directed against a class I antigen that could not be found on target erythrocytes. Sera removed at different stages of pregnancy exhibited varying degrees of cross-reactivity. To provide a source of pregnancy-induced antibody spleens from mice were removed during pregnancy and fused with rat or mouse myelomas. Antibody-secreting hybridomas were sought by means of CELISA with paternal cells as targets. Four hybridomas were isolated, producing antibody of the IgGl subclass, directed against a class I antigen and with limited cross-reactivity. The target antigen for both pregnancy sea and monoclonal antibody was examined for H-2 linkage, using the Lod score. The results obtained were unusual. Combination of the scores for four separate sera suggested an MHC-linked target. Individual scores suggested that two sera were directed against a linked and two against an unlinked antigen. Three of the monoclonal antidbodies were directed against H-2-linked antigens. Both sera and monoclonal antibody were immunoblotted against paternal, maternal and control cells. Pregnancy sera was seen to blot a 45-kD antigen present on paternal strain cells and cells from a mouse sharing the maternal haplotype. Only one hybridoma could be successfully blotted, revealing a 45-kD target. Immunisation with third-party lymphocytes has been used to treat recurrent spontaneous abortion. In twenty two couples treated in this way immunisation proved to be beneficial but there was no evidence for importance of an immune response or HLA sharing.
33
Ueda, Yasuji. "MONOCLONAL ANTIBODIES TO CHICK CRYSTALLINS". 京都大学 (Kyoto University), 1989. http://hdl.handle.net/2433/86412.
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34
Pathan, N. "Catalytic monoclonal antibodies: a review". Thesis(M.Phil.), CSIR-National Chemical Laboratory, Pune, 1990. http://dspace.ncl.res.in:8080/xmlui/handle/20.500.12252/2017.
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35
Haron, Sharifah Zabidah. "Engineering recombinant antibodies for virus resistance". Thesis, University of Leicester, 1997. http://hdl.handle.net/2381/29815.
Testo completoAbstract (sommario):
An important step in the development of plant protection strategies using anti-viral recombinant antibodies will be to devise generic systems for the reliable expression of the antibodies to the required levels in the desired cellular compartment. Currently, expression levels of recombinant antibodies are apparently antibody dependent, highly variable and in the cytoplasmic compartment in particular, often very low. An aim of the work reported here was to investigate the possibility that the fusion of recombinant antibodies to a protein known to be highly expressed in plants would increase the level of the fusion protein over that of the separately expressed recombinant antibody. The potato virus-S virus coat protein accumulates at high levels in the plant and was therefore selected as a fusion partner for two combinant antibodies; a model scFv, AS32scFv, known to be functional and to accumulate, although at low levels, in the cytosol of Nicotiana tabacum; and an scFv antibody to coat protein of potato virus-V, scFv4.5. These gene fusions were constructed and expressed in a protoplast transient expression system and an Agrobacterium rhizogenes hairy root system. The hairy root system, as determined by the se of GUS reporter gene expression, proved to be convenient and reliable. Evidence was obtained with ELISA detection methods which indicates that fusion of scFv to viral coat protein increases scFv accumulation in the cytoplasm although difficulties with the detection of the unfused scFv on western blots prevented thorough investigation. Other anti-viral scFv's were made available for study but attempts to express them in a bacterial system for characterisation purposes were unsuccessful. The Pichia yeast expression system was therefore chosen as a possible alternative means of producing these scFv's, in sufficient quantities for characterisation as scFvs and scFv fusions prior to the labour intensive and time consuming process of plant transformation. Although after considerable effort the system allowed the production of significant amounts of one of the antibodies studied it did not prove possible to detect expression of the two other anti-viral antibody genes investigated.
36
Cao, Ying. "Development and applications of bispecific antibodies". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape7/PQDD_0026/NQ39510.pdf.
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37
Alexandrovich, Susan K. "Characterization of monoclonal antibodies against digoxin /". Online version of thesis, 1987. http://hdl.handle.net/1850/10681.
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38
Mirza, Myriam. "Characterization of new CFTR monoclonal antibodies". Thesis, McGill University, 2009. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=66882.
Testo completoAbstract (sommario):
The available antibodies against CFTR are not sensitive enough to detect CFTR at endogenous or near endogenous levels making detection at native levels difficult. We raised two monoclonal antibodies, 22E8 and 23C5, against the R domain of human CFTR with the goal of identifying an antibody sensitive enough to detect CFTR in native airway cells. These antibodies were characterized for their ability to detect over-expressed as well as endogenous levels of CFTR in immunoblotting, immunoprecipitation and immunofluorescence. Their ability to detect CFTR was also compared with commercial antibodies M3A7 and 24-1. We show that 23C5 and 22E8 are more sensitive than the commercial antibodies and are able to detect CFTR in over-expressed and endogenous cells by immunoblotting. However, only 23C5 is able to immunoprecipitate CFTR and neither is able to detect CFTR in native airway cells by immunoblotting or are suitable for immunofluorescence. These antibodies will enable studies of CFTR biogenesis in endogenous cells.A l'heure actuelle les anticorps dirigés contre la protéine CFTR ne sont pas suffisamment sensibles pour détecter cette protéine de facon endogène rendant ainsi l'étude de cette protéine difficile dans les tissus. Notre laboratoire a fabriqué deux anticorps monoclonaux , nommés 22E8 et 23C5, dirigés contre le domaine R de la protéine CFTR. L'abilité de ces anticorps à détecter l'expression de CFTR que ce soit de façon endogène ou lorsque la protéine est surexprimée a été testée à l'aide des techniques d'immunoblotting, d'immunoprécipitation et d'immunofluorescence. Afin de verifier leur sensibilité et leur capacité à détecter la protéine CFTR, ces anticorps ont été comparés aux anticorps M3A7 et 24-1 qui sont disponibles dans le commerce et connus pour détecter de facon optimale la protéine CFTR. Les resultats obtenus dans les lignées cellulaires à l'aide de la technique d'immunoblotting ont permis de montrer que les anticorps 23C5 et 22E8 sont plus sensibles que les anticorps commerciaux, de plus ils sont capables de détecter à la fois les protéines endogènes et sur-exprimées. Bien que l'anticorps 23C5 soit capable d'immunoprécipiter la protéine CFTR, aucun des deux anticorps n'a permis la detection de la protéine CFTR par immunoblotting dans les cellules de culture primaire. De plus, ces anticorps n'ont pas permis la detection de la protéine CFTR par immunofluorescence. Ainsi l'utilisation de ces anticorps nous donnera l'opportunité d'étudier la protéine CFTR dans les cellules l'exprimant de facon endogène afin de mieux comprendre sa regulation et son traffic.
39
Noble, Philip W. "Characterisation of anti-glycan monoclonal antibodies". Thesis, University of Nottingham, 2011. http://eprints.nottingham.ac.uk/12071/.
Testo completoAbstract (sommario):
The aims of this thesis are to establish the therapeutic value of two anti-glycan mAbs produced in-house, to develop an immunisation protocol with the aim of improving the immunogenicity tumour-associated glycolipids with the intention of producing therapeutically valuable mAbs and to determine the implication of a mAb with the ability to induce apoptosis in colorectal cancer. The anti-glycan mAbs 692/29 and 505/4 have previously been produced in-house and this study aimed to determine their fine specificity using a glycan array. 692/29 displayed binding predominantly to Lewis b as well as Lewis y-containing glycans. 505/4 was discovered to bind to sialyl Lewis a as well as sialyl di-Lewis a, with no cross-reactivity with other blood group antigens. This was compared to other anti-Lewis mAbs, with differences in specificity being observed. Characterisation of 505/4 mAb distribution showed binding to 80% of colorectal tumours and low levels of binding to normal tissues by IHC, suggesting it may be therapeutically useful. This thesis aimed to assess the ability of 505/4 and 692/29 to meditate immune mediated and non-immune mediated cell death as well as to determine whether non-immune-mediated cell death would be a desirable therapeutic property. Resistance to apoptosis is one of the hallmarks of cancer cells and mAbs stimulating apoptosis may not be very effective. Alternatively, cancer cells are driven to initiate apoptosis by genomic and other aberrations thus if pro-apoptotic pathways are stimulated these cells may be more susceptible to death than normal cells. To investigate the significance of apoptosis in cancer a large tissue microarray of colorectal tumours was assessed for apoptosis and its relationship to patient prognosis. Cleaved caspase-3 is a good marker of apoptosis as it is the executioner caspase for both the extrinsic and intrinsic pathways. Immunohistochemical analysis of colorectal tumour samples revealed that a high expression of cleaved caspase-3 in tumour was associated with good prognosis in colorectal cancer. This suggested that some tumours were still susceptible to apoptotic death but some are resistant and an alternative mechanism of cell death may be an advantage in these tumours. High expression of cleaved caspase-3 in the tumour-associated stroma was also an independent marker of good prognosis in colorectal cancer. This may be because apoptosis of the tumour-associated stroma reduces the level of pro-tumour signals originating from tumour-associated immune cells and stromal cells. As the tumour microenvironment can act in an immunosuppressive and pro-tumour manner, the ability of a mAb to induce direct cell death without the need for effector cells or complement would be an advantage. Lewis y and Lewis b are blood group antigens commonly overexpressed on the surface of a range of cancers. Characterisation of effector functions of 505/4 and 692/29 demonstrated that both mAbs have the ability to mediate apoptosis by antibody dependent cellular cytotoxicity, complement dependent cytotoxicity and cause direct cell death in an oncosis-like manner. Comparison with other anti-Lewis mAbs demonstrated that a number of anti-Lewis mAbs can induce direct cell death independently of apoptosis. Thus, they could effectively target apoptotic sensitive and resistant colorectal cancers. Tumours aberrantly express glycolipids and these molecules may be involved in a number of cellular pathways. In addition a large proportion of anti-glycan mAbs, including 505/4 and 692/29 in this thesis, have displayed the ability to induce direct cell death. Therefore this thesis aimed to develop an immunisation protocol capable of increasing the immunogenicity of tumour-associated glycolipid for the production of anti-tumour glycolipid mAbs directed against ovarian cancer. This study suggests that the incorporation of tumour glycolipid into liposomes and their immunisation along with the iNKT cell adjuvant α-galactosylceramide, elicits an anti-tumour glycolipid immune response, which can yield IgG mAbs capable of binding a high proportion of ovarian cancers. In summary, this thesis confirmed specificity of 692/29 to Lewis y and Lewis b and 505/4 to sialyl Lewis a and sialyl di-Lewis a. Furthermore, this thesis demonstrated a promising tissue distribution of 505/4 in vitro. Characterisation of mAb effector functions suggest that both Lewis y and sialyl Lewis a directed mAbs have the ability to cause direct cell death, independently of apoptosis in antigen positive cells, as well as the ability to cause immune-mediated cell death. This may be an important factor in the immune-suppressive tumour microenvironment. Furthermore, this thesis provides the basis for the production of new anti-glycolipid antibodies that may also be able to induce direct cell death.
40
Raghavan, A. K. "Sequence and structural analysis of antibodies". Thesis, University College London (University of London), 2009. http://discovery.ucl.ac.uk/15808/.
Testo completoAbstract (sommario):
The work presented in this thesis focusses on the sequence and structural analysis of antibodies and has fallen into three main areas. First I developed a method to assess how typical an antibody sequence is of the expressed human antibody repertoire. My hypothesis was that the more \humanlike" an antibody sequence is (in other words how typical it is of the expressed human repertoire), the less likely it is to elicit an immune response when used in vivo in humans. In practice, I found that, while the most and least-human sequences generated the lowest and highest anti-antibody reponses in the small available dataset, there was little correlation in between these extremes. Second, I examined the distribution of the packing angles between VH and VL domains of antibodies and whether residues in the interface in uence the packing angle angle. This is an important factor which has essentially been ignored in modelling antibody structures since the packing angle can have a signicant eect on the topography of the combining site. Finding out which interface residues have the greatest in uence is also important in protocols for `humanizing' mouse antibodies to make them more suitable for use in therapy in humans. Third, I developed a method to apply standard Kabat or Chothia numbering schemes to an antibody sequence automatically. In brief, the method uses proles to identify the ends of the framework regions and then lls in the numbers for each section. Benchmarking the performance of this algorithm against annotations in the Kabat database highlighted several errors in the manual annotations in the Kabat database. Based on structural analysis of insertions and deletions in the framework regions of antibodies, I have extended the Chothia numbering scheme to identify the structurally correct positions of insertions and deletions in the framework regions.
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Besarani, Dler. "Anti-Vimentin Antibodies in Renal Transplantation". Thesis, Imperial College London, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.526360.
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Chowdhury, Saifuddin M. Zahed. "Antineutrophil cytoplasmic antibodies and systemic vasculitis". Thesis, University of Newcastle Upon Tyne, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.327199.
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Thanh, Le Thiet. "Exon-specific monoclonal antibodies against dystrophin". Thesis, University of Salford, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.261661.
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Drever, Matthew. "Generating microcystin antibodies by phage display". Thesis, University of Aberdeen, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.445137.
Testo completoAbstract (sommario):
A naïve human antibody fragment phage display library was screened against conjugated microcystin-LR (MCLR). Phage antibodies were eluted with free MCLR to promote the isolation of free antigen binders. Isolated antibody fragments were cloned into a soluble expression vector and single-chain antibody (scAb) expressed in a bacterial host. All scAbs bound free antigen in an indirect competition ELISA and levels of sensitivity were determined. The most responsive clone 3A8 had an 800-fold increase in sensitivity to MCLR than previously documented scAbs and was able to detect levels below guidelines for MCLR in drinking water set by the World Health Organisation (WHO) at 1 μg/L. Despite its sensitivity to MCLR, full characterisation revealed low levels of cross-reactivity with other microcystin variants. In vitro affinity maturation was employed to increase cross-reactivity to other microcystin variants in scab 3A8. A chain-shuffled library was constructed where a naïve VL repertoire was shuffled with clone 3A8 VH chain. The library was screened and isolated phage antibodies cloned and expressed as scAbs. Chain-shuffled clone CSB-D9 displayed a 50-fold increase in cross-reactivity for a single microcystin variant and was capable of detecting all variants available below the guideline levels of 1 μg/L. The highly cross-reactive scab was used for the determination of total microcystin content in cyanobacterial extracts and results showed good correlation with HPLC quantification. Immunoaffinity chromatography utilising scab CSB-D9 was used for the concentration of trace amount of microcystin from large volumes of water, out-performing columns generated with anti-microcystin whole antibody molecules. The ability of scab CSB-D9 to protect cultured hepatocytes against microcystin-induced apoptosis was also determined. Results indicated a possible therapeutic application for human antibody fragments isolated from phage display libraries.
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Watson, Nigel. "Monoclonal antibodies to human immunoglobulin allotypes". Thesis, University of Strathclyde, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.304897.
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Ortlepp, Susan. "Leucocyte integrin activation by monoclonal antibodies". Thesis, Open University, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.359976.
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Holdsworth, Mary Louise. "Characterisation of phytochrome using monoclonal antibodies". Thesis, University of Leicester, 1987. http://hdl.handle.net/2381/35466.
Testo completoAbstract (sommario):
Characterisation of phytochrome using monoclonal antibodies Mary L. Holdsworth Native oat phytochrome has been purified to homogeneity and used to produce a panel of monoclonal antibodies (mAbs). Selection of mAbs followed early screening against native phytochrome by ELISA, and SDS-denatured phytochrome by "mini" western blotting. Six mAbs which recognised SDS-denatured phytochrome were mapped using proteolytically derived fragments of phytochrome and subsequent immunoblotting. LAS 31 and 33 map to the 6 kDa NH2-terminus and LAS 35 and 41 map to the adjacent 4 kDa sub-NH2- terminal domain. LAS 11 maps to the 64 kDa- chromophore-bearing domain and LAS 32 maps to between 74 and 88 kDa from the NH2-terminus on the COOH- terminal-half of the molecule. A novel protocol for the mapping of conformation-specific mAbs has been developed and used to assign LAS 21, 34 and 42 to the 64 kDa-chromophore-bearing domain. Determination of differential affinities towards Pr and Pfr demonstrated that LAS 42 exhibited a higher affinity for Pfr, LAS 31, 33, 34 and 35 exhibited a higher affinity for Pr. LAS 41 discriminates absolutely against Pfr. LAS 41 has therefore facilitated:- (i) the purification of PfrP, i.e. Pfr which is free of contaminating Pr, (ii) the development of an ELISA for phytochrome photoequilibrium, (iii) the first direct experimental evidence that phytochrome can exist as a stable heterodimer in vitro and (iv) an appraisal of ELISA protocols for determining differential affinities of mAbs towards Pr and Pfr. Spectral analyses of phytochrome in the presence of mAbs have underlined the importance of the 6 kDa NH2-terminus in the maintenance of the spectral integrity of the molecule but have also indicated that the 4 kDa sub-NH2-terminal domain also interacts with the chromophore. Cross reactivity studies amongst phytochrome from monocots and dicots demonstrate that the epitopes recognised by LAS 11, 31, 33, 35 and 41 are not highly conserved. However, LAS 32 recognises phytochrome from every plant species tested, and is therefore recognising a highly conserved region on the molecule.
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Ferreira, Filipe Miguel Garcia. "Antibodies purification using centrifugal partition chromatography". Master's thesis, Universidade de Aveiro, 2017. http://hdl.handle.net/10773/22486.
Testo completoAbstract (sommario):
Mestrado em Bioquímica - Métodos BiomolecularesA dificuldade em desenvolver antibióticos mais eficientes, numa altura em que a resistência microbiana tem vindo a aumentar, torna essencial o desenvolvimento de terapias alternativas, económicas e eficazes. Os anticorpos obtidos a partir da gema de ovo de galinha, imunoglobulina Y (IgY), têm-se destacado não só pela sua produção mais simples e em maior quantidade em relação aos anticorpos policlonais de mamífero, mas também devido às inúmeras vantagens em termos de aplicações. No entanto, atualmente não existe uma plataforma de purificação de IgY que seja económica, eficaz e passível de aplicação a nível industrial - uma lacuna que este trabalho se propõe a resolver. Assim, neste trabalho, estudou-se a possibilidade da utilização de sistemas aquosos bifásicos (SAB) compostos por PEG 1000 e tampão fosfato (K2HPO4/KH2PO4) ou K2HPO4, seguidos de um passo de ultrafiltração, ou acoplados à tecnologia de cromatografia de partição de força centrífuga (CPC), para a purificação de IgY. Foram avaliados os efeitos de pH (5,5; 6,0; 6,5; 7,5; e 8,0) e composição de PEG e sal na extração de IgY, bem como as condições utilizadas na CPC (fluxo da fase móvel, rotação e modo de operação). Foi estudada a estabilidade do anticorpo em soluções aquosas dos componentes utilizados nos SAB utilizando dicroísmo circular, assim como a atividade/estabilidade do anticorpo após o processo de purificação por ELISA, salientando assim o efeito do PEG 1000 na estrutura secundária e na atividade do IgY. O ATPS constituído por 18 % PEG 1000 + 13 % tampão fosfato a pH 6,0 conduz aos melhores resultados em termos de purificação, obtendo-se num único passo de extração uma pureza de IgY de 39 %. Após a aplicação de CPR, obteve-se IgY com um grau de pureza de 51 %, e com ultrafiltração, IgY com um grau de pureza de 47 %. Face aos resultados obtidos, destaca-se a CPR como a técnica mais adequada dado que permite obter IgY com um maior grau de pureza e ser passível de aplicação à escala industrial.
The difficulty in developing more effective antibiotics, at a time where the microorganism’s resistance to them has been increasing, turns essential the development of cheaper and effective alternative therapeutics. Antibodies obtained from the chicken’s egg yolk, immunoglobulin Y (IgY), have stood out because of their production simplicity and production in higher quantity when compared to mammal polyclonal antibodies, and also because of their advantages in terms of applicability. Nonetheless, there is still no low-cost, effective and scalable platform for the IgY purification - a vacuity that this work aims to solve. Therefore, in this work, the possibility of using aqueous biphasic systems (ABS) composed of PEG 1000 and phosphate buffer (K2HPO4/KH2PO4) or K2HPO4, followed by an ultrafiltration step, or coupled with centrifugal partition chromatography (CPC), was studied. The effect of pH (5.5; 6.0; 6.5; 7.5 and 8.0) and the PEG + phosphate buffer composition in the extraction of IgY was investigated, as well as the conditions to be used in CPC (mobile phase flow rate, rotation, and the operation mode). The stability of the antibody in aqueous solutions of the components used in the ABS formation was studied using circular dichroism, as well as the activity/stability of the antibody after the purification process, by ELISA, primarily outlining the effect of PEG 1000 in the secondary structure and activity of IgY. The ABS composed of 18 wt % PEG 1000 + 13 wt % phosphate buffer at pH 6.0 yielded the best results in terms of purification, achieving a 39 % IgY purity in a single extraction process. After the application of CPC, an IgY purity of 51 % was obtained, and with the ultrafiltration technique a purity of 47 % was obtained. According to these results, CPC appears as the most adequate purification technique due to the higher purity of IgY obtained and possibility of being applied at an industrial level.
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Koers, Alexander Magnus Maria. "Radiolabelling and biodistribution of IgE antibodies". Thesis, King's College London (University of London), 2015. http://kclpure.kcl.ac.uk/portal/en/theses/radiolabelling-and-biodistribution-of-ige-antibodies(08a7505b-018b-4bf9-a23f-d31b2432d07a).html.
Testo completoAbstract (sommario):
Antibodies used for cancer treatment and immune therapy have, to date, been of the IgG class. Efficacy might be improved by using the IgE class of antibodies as these have higher affinity for their Fc-receptors. IgE has a greater tissue penetration and longer half-life in tissue and IgE bound to IgE-receptor-expressing effector cells is thought to actively infiltrate tumours. IgE has not been subject of in vivo imaging studies to date. Objectives: The aim was to radiolabel both anti-CSPG4-IgE and MOV18-IgE, and their IgG counterparts targeted to the same antigen, while maintaining the functionality of the antibodies; and subsequently, to compare IgE with IgG in in vivo imaging and biodistribution studies in a disease model. Methods: IgE’s and their antigen-matched IgG counterparts were engineered against two different tumour antigens. Tumour models were developed to assess targeting in vivo and imaging and biodistribution studies using radiolabelled IgE were carried out. Six antibodies were engineered for comparison: MOv18-IgE and -IgG antibody targeted against the folate receptor alpha (FR ) expressed on ovarian cancer cells; A MOv18-IgE and -IgG chimeric rat/mouse were engineered for evaluation in a new syngeneic immunocompetent rat model to mimic the immunotherapeutic antibody (human/mouse chimeric MOv18-IgE and -IgG) planned for a clinical study; and a second antibody, anti-CSPG4-IgE, targeted against the chondroitin sulfate proteoglycan 4 (CSPG4), expressed in melanoma cancer was compared to its IgG counterpart. The antibodies were analysed in a NOD/SCID xenograft mouse model with splenic engraftment of human peripheral blood lymphocytes. All antibodies were labelled with 111In using the same bifunctional chelator and labelling conditions (p-SCN-CHX-A"-DTPA) and radiolabelled with 111In at room temperature. Functional assays using FACS were carried out to assure binding to the target and high- and low-affinity Fc-binding to Fc"R expressing immune effector cells. NanoSPECT/CT imaging and biodistribution studies were used to determine targeting and clearance of IgE in vivo. Results: 111In-CHX-A"-DTPA-IgE and -IgG antibodies were labelled with high efficiency (>98%). Binding of the conjugated antibody to the target antigen and Fc"R expressing immune effector cells was identical to that of the native antibody. IgE showed a higher liver uptake in biodistribution (up to 75% ID IgE vs 5% ID IgG 8 h post injection) compared with the IgG counterpart as well as more than 10 times faster blood clearance where as IgG showed prolonged half-life (2.5 days) in blood. Tumour-to-blood and tumour-to-muscle ratios of IgE and IgG showed significant (tbr: P<0.05; tmr: P<0.0005) differences. Conclusion: Similar conjugation and radiolabelling of all antibodies as well as the in vivo assessment allowed the evaluation of targeting, biodistribution and clearance of IgE compared with its IgG counterpart. Observed characteristics of IgE like the rapid blood clearance and the higher liver uptake were found to be fundamentally different compared to its IgG counterpart and suggest a different and mostly unknown mode of action.
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Giorno, Caterina [Verfasser]. "Glycoengineering of Monoclonal Antibodies / Caterina Giorno". Konstanz : Bibliothek der Universität Konstanz, 2010. http://d-nb.info/1020366117/34.
Testo completo