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1
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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Xu, Wenbin. "Studies of antigenic relationships among spotted fever group rickettsiae by monoclonal antibodies". Aix-Marseille 2, 1997. http://www.theses.fr/1997AIX20665.
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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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Fernandes, Carla Sofia. "Análise retrospectiva do achado de pico monoclonal em proteinogramas de rotina". Master's thesis, Universidade da Beira Interior, 2010. http://hdl.handle.net/10400.6/770.
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Introdução: As gamapatias monoclonais constituem um grupo heterogéneo de patologias, caracterizado pela proliferação monoclonal de plasmócitos que produzem e secretam imunoglobulina ou fragmentos desta. Na maioria das vezes, trata-se de uma entidade benigna, usualmente referida como gamapatia monoclonal de significado indeterminado. Contudo, esta pode evoluir para uma situação mais grave como o mieloma múltiplo ou outras gamapatias malignas. A detecção de componente monoclonal através de electroforese e a identificação por imunofixação sérica e/ou urinária de rotina são fundamentais para o seu diagnóstico, dada a sua variabilidade de manifestações clínicas. O principal objectivo deste estudo é salientar a importância da componente laboratorial no diagnóstico das gamapatias monoclonais.
Métodos: Efectuou-se uma análise retrospectiva das electroforeses e imunofixações realizadas no Serviço de Patologia Clínica do Centro Hospitalar Cova da Beira durante o ano de 2009.
Resultados: Neste estudo, foram incluídos 3407 indivíduos, 1983 (58,2%) do sexo feminino e 1424 (41,8%) do sexo masculino, com uma idade média de 65 anos. Durante o ano de 2009, a incidência de picos monoclonais foi de 3,55%. Os serviços de Hematologia e de Medicina Interna foram os que detectaram o maior número de picos monoclonais. Do total de indivíduos com componente monoclonal, 74 (61,2%) eram do sexo masculino e 47 (38,8%) do sexo feminino, apresentando uma idade média de 72 anos. A incidência das cadeias pesadas foi de 59,5% para IgG, 22,4% para IgM e por último 15,6% para IgA. Em relação às cadeias leves, a incidência de kappa foi de 62% e de lambda 38%. Em 5 indivíduos foram detectados picos biclonais, tendo-se obtido uma incidência de 4,13%.
Conclusão: A electroforese de proteínas pode ser considerada um bom método de triagem para detecção precoce de gamapatias monoclonais e a imunofixação para confirmação diagnóstica e para a caracterização das gamapatias monoclonais.Introduction: Monoclonal gammopathies comprise of a heterogeneous group of pathologies, characterized by the monoclonal proliferation of plasmocytes, which produce and secrete immunoglobulins or fragments of these. In the majority of cases, this is a benign entity known as monoclonal gammopathy of undetermined significance. However, it is possible that these gammopathies may lead to the development of more serious conditions, mainly multiple myeloma and other malignant gammopathies. Critical to the diagnosis of these immunoglobinopathies is the routine detection of the monoclonal component by means of electrophoresis and serous/urinary immunofixation, as they may present with a wide variety of clinical manifestations. The main objective of this study is to emphasize the importance of laboratorial screening in the diagnosis of monoclonal gammopathies. Methods: A retrospective study was carried out, consisting in the analysis of electrophoresis and immunofixations applied to patients of the Clinical Pathology Department of the Cova da Beira Hospital Centre throughout the year 2009. Results: This study comprised of 3407 participants, 1983 (58.2%) females and 1424 (41.8%) males, with an average age of 65 years. Throughout the year 2009, the incidence of monoclonal peaks pregistered at the Cova da Beira Hospital Centre was 3.55%. The departments which detected the largest number of monoclonal peaks were Hematology and Internal Medicine. Within the total number of individuals presenting with monoclonal components, 74 (61.2%) were male and 47 (38.8%) were female, with an average age of 72 years. The incidence of heavy chains was 59.5% for IgG, 22.4% for IgM and 15.6 % for IgA. With respect to the light chains, the incidence of kappa chains was 62% and lambda chains 38%. Biclonal peaks were found in 5 individuals with a corresponding incidence of 4.13%. Conclusion: The electrophoresis of proteins may be considered as a good screening method for the early detection of monoclonal gammopathies, and immunofixation can be employed for confirmation of the diagnosis and characterization of the monoclonal gammopathies.
5
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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6
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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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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8
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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9
Paudel, Subhash. "Shear thinning in monoclonal antibodies". Thesis, Kansas State University, 2016. http://hdl.handle.net/2097/32833.
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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.
10
Ueda, Yasuji. "MONOCLONAL ANTIBODIES TO CHICK CRYSTALLINS". 京都大学 (Kyoto University), 1989. http://hdl.handle.net/2433/86412.
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11
Merlet, Véronique. "Les anticorps monoclonaux en imagerie médicale : application en cancérologie". Paris 5, 1989. http://www.theses.fr/1989PA05P133.
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Alexandrovich, Susan K. "Characterization of monoclonal antibodies against digoxin /". Online version of thesis, 1987. http://hdl.handle.net/1850/10681.
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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.
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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.
14
Noble, Philip W. "Characterisation of anti-glycan monoclonal antibodies". Thesis, University of Nottingham, 2011. http://eprints.nottingham.ac.uk/12071/.
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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.
15
Coles, A. "Monoclonal antibody therapy of multiple sclerosis". Thesis, University of Cambridge, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.597844.
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T cells mediate the inflammatory activity of multiple sclerosis, which is directed against an unknown autoantigen. A non-antigen specific treatment strategy was tested, drawing on the experimental demonstration of long term allograft acceptance following short term therapy with monoclonal antibodies against T cells. The treatment of 27 patients with multiple sclerosis using a single pulse of humanised anti-lymphocyte (CD52) antibody, Campath-1H, was investigated. With the first dose of monoclonal antibody, patients experienced a rehearsal of previous relapses that fully resolved and was associated with a transient rise in serum TNF-α and IFN-γ. This suggested that inflammatory mediators may impede axonal conduction at previously demyelinated sites. After five consecutive daily doses of Campath-1H, the circulating T lymphocyte count was suppressed for at least 18 months, without any serious infective complications. The in vitro mitogen induced proliferation, and IFN-γ secretion, of patients' peripheral blood mononuclear cells was reduced after treatment and there was also a signficant rise in the peripheral B cell count above pre-treatment levels. This deviation of immune responses away from the Th1 phenotype would, under the hypothesis that multiple sclerosis is a "Th1 disease", be expected to abrogate cerebal inflammation. Unexpectedly, one third of patients developed Graves' disease, an adverse effect not induced by any other therapies nor by Campath-1H treatment of other diseases. By analogy with experimental models of autoimmunity following prolonged lymphocyte depletion, Campath-1H may have selectively depleted T cell clones which suppress autoreactive T cells, although why Graves' disease specifically was induced remains unexplained. Radiological markers of cerebral inflammation were suppressed for at least 18 months in all patients, who experienced no new relapses. However half the patients continued to accumulate disability from deficits acquired prior to monoclonal antibody treatment. In these patients there was both a higher inflammatory load at baseline and progressive cerebral atrophy, which may represent axonal degeneration. It is concluded that inflammation is a necessary prerequisite for new lesion formation and that the secondary progressive phase of multiple sclerosis is initiated by demyelination but proceeds by non-inflammatory mechanisms.
16
Hills, Anna E. "Control of monoclonal antibody N-glycosylation". Thesis, University of Kent, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.344101.
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17
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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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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Simpson, Christina M. (Christina Margaret). "Cost modeling for monoclonal antibody manufacturing". Thesis, Massachusetts Institute of Technology, 2011. http://hdl.handle.net/1721.1/66050.
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Thesis (M.B.A.)--Massachusetts Institute of Technology, Sloan School of Management; and, (S.M.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering; in conjunction with the Leaders for Global Operations Program at MIT, 2011.Cataloged from PDF version of thesis.
Includes bibliographical references (p. 75-76).
The Novartis BioPharmOps division is responsible for manufacturing large molecule products, including monoclonal antibodies, for late stage clinical trials and commercial sales. The BioPharmOps site in Huningue, France is expanding their product line but is also trying to reduce costs; cost pressures are increasing as biotech products become a larger part of Novartis' pipeline. The site uses a standard cost method to calculate their product costs. However, when using standard costs it can be time-consuming to extrapolate and predict costs when inputs and assumptions (such as product mix or process parameters) are changed. This project describes development of a model that allows the factory to quickly and easily simulate new product mixes and process flows. This model provides the site with a different view of their costs that will help them understand their cost drivers more completely and thereby help enable strategic decision-making at the site. A model of this type can be used to provide unexpected insights but the data in it are not meant to stand alone. By using results from a cost model like this along with operational metrics like throughput time or changeover time, a site should be able to quickly predict the cost impact of process changes or changes in the production plan.
by Christina M. Simpson.
S.M.
M.B.A.
21
Qian, Qi. "Intracellular delivery of rabbit monoclonal antibody". Scholarly Commons, 2007. https://scholarlycommons.pacific.edu/uop_etds/679.
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In the past decades, a series of small peptides, Protein Transduction Domain (PTD), were discovered to be able to facilitate the delivery of small proteins into living cells. With the specific feature, researchers have successfully delivered some functional proteins into living cells. To fully explore and understand the functions and structures of intracellular proteins, more powerful tools are under demand. Recently, an increasing number of rabbit monoclonal antibodies (RabMAbs) have been approved to able to recognize subtle distinctions between the changes of intracellular proteins status. They could be good tools for researchers with the ability to traverse through cell membrane into living cells.
In this dissertation, a novel delivery technology for RabMAbs was established. Transcriptional activator of transcription (TAT) peptide was utilized as a delivery carrier for RabMAbs. It was demonstrated that RabMAbs could be delivered into living cells by conjugating with TAT peptide. Different cell lines, including adherent and suspension cells, were tested for the delivery of RabMAbs. The delivery process was studied in terms of incubation concentration and time, and an optimal delivery condition was established.
To investigate the biological function of delivered RabMAbs inside cytoplasm, three RabMAbs against actin, procaspase-3 and NF-κB respectively were studied. Their binding activities after delivery were verified via sandwich-ELISA data. The immunofluorescent staining of the delivered RabMAb against actin showed it specifically bound to the actin filament in its native morphology. The quantitative analysis of the delivered RabMAb against procaspase-3 showed that approximately 60% of delivered antibody bound to the antigen proteins. The delivered RabMAb against NF-KB apparently blocked the nuclear translocation of NF-KB introduced by TNF-a. The success of delivering the three rabbit monoclonal antibodies with binding or inhibiting functions demonstrated the feasibility of delivering various RabMAbs into living cells by TAT peptide for studying the biological functions of intracellular proteins.
Furthermore, to overcome the efficiency and cost issues of the RabMAb delivery system, a universal delivery platform for RabMAbs was developed. This platform uses goat-anti-rabbit polyclonal antibody conjugated with TAT peptide as delivery vehicle. It was confirmed that the goat-anti-rabbit polyclonal antibody modified with TAT peptide was able to capture RabMAbs and deliver RabMAbs into living cells by the conjugated TAT peptide. The results provide a promising delivery platform for all RabMAbs.
22
Holdsworth, Mary Louise. "Characterisation of phytochrome using monoclonal antibodies". Thesis, University of Leicester, 1987. http://hdl.handle.net/2381/35466.
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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.
23
Molnar, Steven Albert. "Monoclonal antibody studies of cytochrome F /". The Ohio State University, 1990. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487685204970274.
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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.
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Hammaker, Deepa Rajan. "Monoclonal antibody therapy of rheumatoid arthritis". Diss., The University of Arizona, 1999. http://hdl.handle.net/10150/289074.
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Objectives. To (a) determine the immunological effects of a PRIMATIZED® anti-CD4 antibody alone or in combination with methotrexate in RA patients, (b) determine the immunological effects of a chimeric anti-CD25 antibody in RA patients who are partially refractive to methotrexate and (c) compare interleukin-15 levels in the serum of RA patients and healthy controls and determine if there is a correlation between this cytokine and serum TNF-α, CD 122 expression, and disease activity. Patients and methods. (a) Eight RA patients were selected, four received anti-CD4+ placebo and the other four received anti-CD4+ methotrexate for 4 weeks. The immunological effects were assessed on peripheral blood by flow cytometry and thymidine incorporation assays. (b) Six RA patients were given anti-CD25 antibody (0.02-60mg) along with methotrexate for 26 days. The immunological effects were assessed on peripheral blood by flow cytometry, thymidine assays, and ELISA. (c) Blood and disease activity from twenty-one RA patients were obtained and serum IL-15 and TNF-α levels were measured by ELISA. IL-15R β chain (CD 122) expression was measured by flow cytometry. Results. (a) The anti-CD4 antibody caused a selective and significant decrease in the number of CD4+ T cells. No inhibition of PHA or mitogenic antibody stimulated proliferation was observed. (b) The anti-CD25 antibody caused a significant decrease in the percent CD25+ cells. The antibody bound CD25 and prevented interaction of IL-2 and IL-2R. Anti-CD25 antibody caused a significant decrease in PHA or mitogenic antibody stimulated proliferation. Clinically, the anti-CD25 antibody caused a significant decrease in the number of tender and swollen joints. (c) Elevated serum IL-15 was measured in 10 out of 21 RA patients but not in controls. No correlation was observed between IL-15 and TNF-α, CD122 expression or disease activity. Conclusions. (a) Methotrexate did not alter the effects of the PRIMATIZED® anti-CD4 antibody. Changes in antibody development processes have yielded two antibodies with different functions. (b) Anti-CD25 induced decrease in CD25+ T cells was associated with clinical benefit. The exact mechanisms of action are not clear. (c) Serum IL-15 levels in RA may be a more sensitive indicator of inflammation than TNF-α and may be a valuable tool in diagnosis.
26
Yeung, Douglas Edward. "Characterization of six monoclonal antibodies against the Minute Virus of Mice NS-1 protein, and the use of one in the immunoaffinity purification of NS-1 expressed in insect cells". Thesis, University of British Columbia, 1990. http://hdl.handle.net/2429/29405.
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Six mouse monoclonal antibodies have been isolated which react against a bacterial fusion protein containing amino acids 364 to 623 of the NS-1 protein of the prototype strain of the Minute Virus of Mice (MVMp). All six were found to be of the IgG class of antibodies; five being IgG₁ and the sixth being IgG₂[formula omitted]. By immunoblot analyses, these antibodies all recognize an 83 kDa protein found only in MVM-infected mouse fibroblast cells, leading to the assumption that they are all NS-1 specific. Further evidence for this assumption is obtained from indirect immunofluorescence studies showing all but one of the mAbs react against a nuclear protein found in MVM-infected cells.
The epitopes of the antibodies were mapped using carboxy-terminal deleted bacterial fusion proteins derived from the plasmid encoding the original antigen. For the six monoclonal antibodies, four distinct epitopes were found (A - D). Three were clustered in a 16 amino acid region near the carboxy-terminal of the bacterial fusion protein, while the fourth was slightly more toward the amino-terminal side. Competition ELISAs against a 25 amino acid NS-1 specific peptide confirmed the mapping of the A epitope recognized by the CE10 and AC6 monoclonal antibodies.
Also in this thesis, the characterization of a NS-1 fusion protein and a non-fused NS-1 protein expressed in insect cells by recombinant baculoviruses is also described. The latter, a full-length NS-1 protein designated NS-1[formula omitted]ⅽ, was found to be an 84 kDa cytoplasmic protein. This protein was immunoprecipitated by all six monoclonal antibodies. A CE10 monoclonal antibody immunoaffinity column was employed in the single-step purification of NS-1 [formula omitted]c from insect cells. Four elution methods (alkaline, peptide, 6M guanidinium, and acid) were examined and the best purification was obtained using the acid elution.Medicine, Faculty of
Biochemistry and Molecular Biology, Department of
Graduate
27
Ohlin, Mats. "Human monoclonal antibody technology a tool to investigate human antibody repertoires /". Lund : Dept. of Immunotechnology, Lund University, 1992. http://catalog.hathitrust.org/api/volumes/oclc/39693827.html.
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Pantel, Jacques. "Etude des régions d'interaction entre l'hormone chorionique gonadotrope humaine et son récepteur". Paris 5, 1995. http://www.theses.fr/1995PA05P048.
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Peigue-Lafeuille, Hélène. "Différenciation intratypique et variation antigénique des entérovirus : étude des échovirus en prenant pour modèle l'échovirus type 25". Lyon 1, 1991. http://www.theses.fr/1991LYO1H183.
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Chen, Desheng, e chen desheng@deakin edu au. "Development of monoclonal antibodies against Vibrio pathogens". Deakin University. Department of Biological Science, 1991. http://tux.lib.deakin.edu.au./adt-VDU/public/adt-VDU20080626.140825.
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Monoclonal antibodies were developed against pathogenic vibrios for use in rapid identification in disease situations of humans, fish and shellfish. Of the 12 fusions performed using V. alginolyticus, V. anguillarum, V. carchariae, V. cholerae, V. damsela, V. furnissii, V. harveyi, V. ordalii, V. parahaemolyticus and V. vulnificus, a total of 102 hybridomas were obtained. Based on cross-reactivity of a wide range of Vibrio strains and other gram-negative bacteria, three broad types of monoclonal antibodies were found. The three categories were: (1) ones that were species-specific or specific to a particular surface antigen, (2) a large number that reacted with several Vibrio species, and (3) three that reacted with most Vibrio strains but no other gram-negative bacteria.
Each species-specific monoclonal antibody only recognized its corresponding Vibrio species and was used for identifying unknown species, confirming diagnosis of clinical isolates. In addition, several monoclonal antibodies only cross-reacted with similar Vibrio species, e.g. V. parahaemolyticus and V. alginolyticus which share a common H-antigen. Monoclonal antibodies reacting with several Vibrio species were not of particular use in diagnostic situations. Three monoclonal antibodies of the last group did not react with other genera of the family Vibrionaceae, namely Aeromonas, Photobacterium and Plesiomonas nor a wide range of gram-negative enteric bacteria. These data indicated the existence of an antigenic surface determinant common to Vibrio species. One monoclonal reacted with the heat-stable antigenic determinants on the cell surface as v as lipopolysaccharide extracted from all the vibrios studied, thus making it useful for large- scale screening of acute infections of vibrios.
In a blind test, seven Vibrio species, isolated from 6 marine and a freshwater source were identified by two laboratories using phenetic tests. Results of immunotyping using monoclonals, three of seven were diagnosed as the same species, another three were designated as Vibrio species but could not be classified further due to the library not having the corresponding monoclonal, and one was diagnostically questionable. Two further tests were carried out. An unknown Vibrio formalin-fixed isolated from diseased marine animal was identified as
V. parahaemolyticus by ELISA and FITC. Clinical human isolates of V. alginolyticus, V. parahaemolyticus and V. vulnificus were confirmed by monoclonals.
Australian isolates of V. anguillarum appeared to be mostly of serotype O1. monoclonals raised to V. anguillarum AFHRL 1 reacted with only serotype O1 from Denmark but also most Australian isolates.
All vibrios pathogenic to fish and shellfish, i.e. V. anguillarum, V. ordalii, V. alginolyticus, V. carchariae, V. cholerae, V. damsela, V. harveyi, V. parahaemolyticus and V. vulnificus, were used for attachment studies to fish cells using phase contrast and FITC-immunofluorescence microscopy. Of these vibrios, V. anguillarum, V. ordalii and V. perahaemolyticus, were found to adhere to different cells and tissues of rainbow trout while others did not appear to attach. However, attachment was inhibited by monoclonal antibodies specific to only these three vibrios.
Lipopolysaccharide is well known as being a contributing factor in pathogenicity of gram-negative bacteria. PAGE electrophoresis of extracted LPS from 9 strains covering 6 Vibrio species showed the presence of a common 15,000 D fragment. This fragment was verified by immunoblotting with a genus-specific monoclonal antibody (i.e. F11P411F) recognizing nearly all vibrios. The common LPS fragment was separated and used to raise polyclonal antisera in mouse which reacted strongly with LPS itself, live as well as sodium azide-killed vibrios, but not with other gram-negative bacteria. This raised the possibility of developing vaccine from Vibrio LPS.
Monoclonal antibodies developed in the present study enabled rapid identification of a number of pathogenic Vibrio species. There is still further work to produce monoclonal antibodies against additional vibrios that are probably pathogenic. These included V. fluvialis, V. hollisae, V. metschnikovii, V. minicus, V. salmonella and V. tubiashii. Together the application will be of significance in clinical diagnostic work, in the monitoring of vibriosis in fish farms and in quarantine.
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Bell, Ian Martin. "Monoclonal antibodies as catalysts for cationic cyclisations". Thesis, University of Cambridge, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.387041.
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Storey, E. "Monoclonal antibodies to merozoites of Plasmodium falciparum". Thesis, University of Oxford, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.371577.
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Banbury, David N. "New monoclonal antibodies to visualise vesicular compartments". Thesis, University of Oxford, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.259782.
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Klutz, Stephan [Verfasser]. "Continuous processing of monoclonal antibodies / Stephan Klutz". München : Verlag Dr. Hut, 2016. http://d-nb.info/1115549731/34.
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Honey, C. R. "Immunosuppression with monoclonal antibodies in neural transplantation". Thesis, University of Oxford, 1990. http://ora.ox.ac.uk/objects/uuid:ea39dc7a-4ada-4c21-8cef-4649cb322646.
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Huang, Ling. "Investigation of soya globulins using monoclonal antibodies". Thesis, University of East Anglia, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.302022.
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Carter, J. M. "Monoclonal antibody probes of legume storage proteins". Thesis, University of East Anglia, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.384593.
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James, Marian. "Monoclonal antibody studies of dystrophin and utrophin". Thesis, University of Salford, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.360455.
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Slupsky, Joseph R. "Mechanisms of monoclonal antibody-induced platelet activation". Thesis, University of Liverpool, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.240868.
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Anderson, J. "Investigations on bluetongue virus using monoclonal antibodies". Thesis, Open University, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.374892.
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The main aim of the thesis was the production and characterisation of monoclonal antibodies (Mabs) against bluetongue virus (BTV) and the application of such antibodies to the improvement of BTV diagnosis. The suitability of the indirect ELISA for the detection of antibodies to BTV was examined along with the published protocols for the purification of BTV ELISA antigen. When sera were examined from animals which had experienced either BTV vaccine or any other tissue-culture derived vaccine, host-cell protein contaminants in the BTV ELISA antigen reacted with anti-BHK cell antibodies to give false positive results. None of the published protocols completely removed host-cell protein contaminants. As a solution to these problems monoclonal antibodies were produced to BTV and characterised as to their reactivity against a panel of BTV antigen preparations. They were further characterised by competitive binding assays, and SDS-PAGE analysis of the various BTV preparations with which they reacted optimally. One Mab (3-17-A3), directed against BTV p7, proved suitable for use in a blocking ELISA for the detection of antibodies to all 22 serotypes of BTV. The blocking ELISA proved both more sensitive and more specific than the currently used agar gel precipitin test, and has been adopted as the screening test before importation of semen and embryos into the United Kingdom. A further group of antibodies (characterised by Mab F58) directed against BTV polypeptide NS1, used in a blocking assay detected anti-BTV tubule antibodies. This allows detection of BTV replication in infected animals, and may be used to discriminate between BTV infected animals and animals vaccinated with inactivated BTV vaccine. This work demonstrated the previously unreported BTV-specific nature of BTV NS1. Serum-free suspension culture of the hybridomas and purification of the Mab from tissue-culture supernatant was also developed.
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Jones, Christine Ann. "Monoclonal antibodies in the study of neuropeptides". Thesis, Imperial College London, 1991. http://hdl.handle.net/10044/1/46848.
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Eastwood, David Geoffrey Douglas. "Immunotoxicology of the therapeutic monoclonal antibody TGN1412". Thesis, St George's, University of London, 2015. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.676898.
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Having passed all pre-clinical safety testing, the superagonistic anti -CD28 therapeutic monoclonal antibody (mAb ) TGN 1412, intended for the treatment of rheumatoid arthritis and B-cell chronic lymphocytic leukaemia, was approved by German and UK regulatory authorities for first-in-man Phase One clinical trial. Shortly after infusion, all six healthy trial volunteers suffered unexpected and profound systemic pro-inflammatory cytokine release, later termed a 'cytokine storm,' causing multi-organ failure. This unexpected and near fatal cytokine release syndrome (CRS) publically highlighted the failure of current pre-clinical safety testing procedures, emphasising an urgent need for novel cytokine release assays (CRAs) capable of predicting adverse properties of therapeutic mAbs. A wet coat mAb immobilisation approach, developed here, has proven predictive of clinical outcome and would have anticipated TGN1412 immunotoxicity in man. This approach IS now being widely applied by the pharmaceutical industry and contract research organisations (CROs). Comparative studies, testing TGN 1412 against a panel , of therapeutic mAbs, identified a unique mechanism of TGNl412-driven cytokine release. Substantial concentrations of the cytokine lL-2 were subsequently found to be a hallmark for the cytokine storm observed in-vivo, signifYing IL-2 as a TGN1412-like response biomarker. Multiple pro-inflammatory cytokine release was also shown to be principally effector memory T cell (T EM) derived in man. Human and macaque comparative immunophenotyping crucially identified macaque T EM cells as lacking CD28 expression, explaining pre-clinical animal model testing failures. A more physiologically relevant aqueous phase co-culture assay using monocyte-derived dendritic cells is also shown capable of eliciting a TGN1412-like cytokine release profile equivalent to that detected in-vivo and in-vitro using wet coat immobilisation; implying presentation in-vivo likely involved dendritic cells. This thesis describes the most likely mechanisms of action responsible for TGN1412 immunotoxicology in man and provides a plausible explanation for the pre-clinical safety testing failures, findings vital to the fields of immunomodulatory therapeutic mAb development and immunotoxicology.
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Catalão, Dianne Marie Barroso. "Caracterização funcional do anticorpo monoclonal humano BH1". Master's thesis, Universidade de Aveiro, 2008. http://hdl.handle.net/10773/802.
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Mestrado em Biologia Molecular e CelularEmbora, existam novas terapias e avanços no tratamento do cancro, por vezes, os benefícios esperados não são alcançados. O drama humano de quem vive diariamente com esta doença, o alto custo, económico e sociall comprova a pertinência em desenvolver novos estudos para encontrar terapias alternativas, eficazes e inovadoras para o tratamento das doenças neoplásicas, como os Anticorpos Monoclonais Humanos. Moléculas HLA classe II têm sido consideradas como uma boa molécula-alvo para o uso na imunoterapia, devido à sua alta expressão em algumas células de leucemia e linfoma. Recentemente, alguns anticorpos monoclonais anti- HLA classe II, foram desenvolvidos, como Hu1D10 (humanizado) e Ch-Lym1 (quimérico). O grupo de Imunologia da Universidade de Vigo (Espanha), tem obtido vários anticorpos Monoclonais Humanos usando ratos transgénicos, como o anticorpo BH1 que reconhece a molécula HLA-II. Este anticorpo actua muito eficientemente na presença de complemento, frente a células tumorais, matando-as, o que sugere que ele poderia ser um potencial agente no tratamento de várias doenças malignas. O principal objectivo do estudo foi determinar se o anticorpo BH1 poderia activar outros mecanismos anti-tumorais, principalmente modificar o crescimento celular e activar a fagocitose. Os nossos resultados indicam que o efeito de BH1 sobre linhas de células B, originam um crescimento tumoral diferente de outros anti-anticorpos HLA classe II (Ch-Lym1) e a capacidade de BH1 para activar a fagocitose "in vitro" é menor do que a capacidade do Ch-Lym1. Portanto, sugerimos a mudança do isótipo IgM do BH1 para um isótipo IgG humano que poderá melhorar a sua aplicação em terapias humanas. ABSTRACT: Although, there are new therapies and advances in the cancer treatment, sometimes the benefits expected are not produced. The human drama who lives daily with this disease, the high cost, economic and social; proves the evidence to develop new studies to find alternative, effective and innovative therapies to the neoplasic diseases treatment, like Human Monoclonal Antibodies. HLA class II molecules have been considered as a good target molecule for use in immunotherapy because of their high expression in some leukemia and lymphoma cells. Recently, some monoclonal antibodies anti-HLA class II have been developed, like Hu1D10 (humanized) and Ch-Lym1 (chimeric). The Immunology group of University of Vigo (Spain) has obtained several Human monoclonal antibodies using transgenic mice, like BH1 that recognizes the molecule HLA-II. This antibody kills very efficiently tumour cells in presence of human complement, suggesting that it could be a potential agent in the treatment of several malignancies. The main objective of our study was to determine if BH1 antibody could activate other anti-tumour activities, mainly modify the cellular growth and activate phagocytosis. Our results indicate that the effect of BH1 on tumoral B cell lines growth is different to other anti-HLA class II antibodies (Ch-Lym1) and the ability of BH1 to activate phagocytosis “in vitro” is lower than the Ch-Lym1 activity. So, we suggest that the isotype changing of BH1 to human IgG could improve its application in human therapy.
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Koch, Tyree J. "Aggregation Propensity: Characterization of Monoclonal Antibody Stability". Thesis, Harvard University, 2015. http://nrs.harvard.edu/urn-3:HUL.InstRepos:24078351.
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The study of aggregation propensity of a monoclonal antibody (mAb) and its sensitivity to applied stresses is believed to correlate with the overall stability of the mAb. As such, the aggregation propensity under various stresses can be used to develop a unique aggregation metric to rank order a panel of mAbs based on their stability. Often in a drug discovery campaign, multiple mAbs may imbue the desired in vivo efficacy, at which point identification of the most developable mAb becomes an important factor to decide on a single candidate for further development. This study focuses on the assessment of the stability of a panel of mAbs, by defining their propensity for aggregation along the native and non-native aggregation pathways. Kosmotrope based solubility evaluates a mAb’s colloidal stability, or propensity for native aggregation, while differential scanning fluorescence reports conformational stability, or propensity for non-native aggregation. By combining the conformational and colloidal stability metrics, an overall aggregation propensity profile can be generated for a mAb. To parse out further information on stability, the mAb panel was exposed to a series of stresses, which mimic stresses a mAb based drug would be exposed to during manufacturing and storage. After exposure to stress, the mAb panel was then monitored for change in apparent colloidal and conformational stability. There was no variation in the stability metrics measured, as a function of stress. However, observed precipitation denoted differential sensitivity to the stresses. Combining observational data with the stability metrics measured, allowed for rank ordering of aggregation propensity, and overall stability.
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Menezes, Márcio Anunciação. "Anticorpos anti-intimina: análise da reatividade dos anticorpos policlonal e monoclonal, clonagem e expressão do fragmento variável de cadeia simples (scFv) do anticorpo monoclonal". Universidade de São Paulo, 2010. http://www.teses.usp.br/teses/disponiveis/9/9136/tde-19032010-161924/.
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Intimina é o principal fator de virulência envolvido na patogênese de Escherichia coli enteropatogênica (EPEC) e de Escherichia coli enterohemorrágica (EHEC). A detecção de EHEC e EPEC típica ou atípica é de fundamental importância na definição da conduta terapêutica das infecções promovidas por E. coli, que ainda são a principal causa de diarreia aguda em crianças e adultos em muitos países desenvolvidos e em desenvolvimento. Anticorpos são ferramentas importantes na detecção de diversos patógenos. Neste trabalho avaliou-se a sensibilidade e especificidade dos anticorpos policlonal e monoclonal anti-intimina frente a isolados de EPEC e EHEC por immunoblotting. Os anticorpos apresentaram 100% de especificidade e a sensibilidade foi de 97%, 92% e 78%, quando se utilizou a fração enriquecida em IgG do soro de coelho, antissoro de rato e anticorpo monoclonal, respectivamente. Esse anticorpo monoclonal anti-intimina foi caracterizado como IgG2b e 1 µg desse anticorpo reconheceu 0,6 µg de intimina purificada com uma constante de dissociação de 1.3 x 10-8 M. A menor reatividade do anticorpo monoclonal em relação aos anticorpos policlonais levou-nos à clonagem e expressão do fragmento variável de cadeia simples desse anticorpo (scFv). Para isso, o mRNA do hibridoma anti-intimina foi extraído, reversamente transcrito para cDNA e amplificadas as cadeias leve e pesada da fração variável do anticorpo, utilizando iniciadores aleatórios comerciais. As cadeias amplificadas foram ligadas ao vetor pGEM-T Easy e sequenciadas. Iniciadores específicos foram desenhados e utilizados em uma estratégia de amplificação e união das cadeias, formando o scFv, que por sua vez foi clonado no vetor de expressão pAE. Linhagem de E. coli BL21(DE3)pLys foi transformada com o plasmídeo pAE-scFv antiintimina e submetida à indução protéica. O scFv anti-intimina foi expresso de forma insolúvel, solubilizado, purificado e submetido ao ensaio de refolding. O rendimento obtido foi de 1 mg de proteína por 100 mL de cultivo bacteriano. Para testar a funcionalidade do scFv, foram realizados ensaios de ELISA de captura e imunofluorescência. Os resultados mostraram que 275 ng de scFv reagiram com 2 µg de intimina purificada a uma absorbância de aproximadamente 0,75 e por imunofluorescência mostrou uma forte reatividade ao isolado de EPEC típica E2348/69. Este estudo demonstrou que o anticorpo recombinante anti-intimina obtido foi capaz de reconhecer a região conservada de intimina (Int388-667) na forma purificada e a intimina α no isolado de EPEC típica, e se mostrou mais eficiente que o anticorpo monoclonal nativo.Intimin is the major virulence factor involved in the pathogenesis of enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic Escherichia coli (EHEC). The detection of EHEC and typical or atypical EPEC has fundamental importance in defining the therapeutic management of infections caused by E. coli, which are still the leading cause of acute diarrhea in children and adults in many developed and developing countries. Antibodies are important tools in the detection of several pathogens. In this study it was evaluated the sensitivity and specificity of polyclonal and monoclonal antibodies against intimin in the detection of EPEC and EHEC by immunoblotting. All employed antibodies showed 100% specificity and the sensitivity was 97%, 92% and 78% for rabbit anti-intimin IgG-enriched fraction, rat antisera and monoclonal antibody, respectively. This anti-intimin monoclonal was characterized as IgG2b and 1 mg recognized 0.6 µg of purified intimin with a dissociation constant of 1.3 x 10-8 M. The less extent reactivity of monoclonal led us to clone and express the single chain fragment variable of this antibody (scFv). Thus, the anti-intimin hybridoma mRNA was extracted, reverse transcribed to cDNA and the light and heavy chains of variable fragment of the antibody were amplified using commercial random primers. The chains were amplified, ligated to the pGEM-T Easy vector and the insert was sequenced. Specific primers were designed and used in a strategy to amplify and link the chains, obtaining the scFv, which was cloned into the pAE expression vector. E. coli BL21(DE3)plys was transformed with the pAE-scFv anti-intimin plasmid and subjected to induction of protein expression. The scFv anti-intimin, expressed in the insoluble fraction, was purified and submitted to refolding. The yield was 1 mg of protein per 100 mL of bacterial culture. To test the functionality of the scFv, ELISA and immunofluorescence assays were performed. The results showed that 275 ng of scFv reacted with 2 µg of purified intimin resulting in an absorbance of 0.75. By immunofluorescence it was observed a strong reactivity to the typical EPEC isolate E2348/69. This study demonstrated that the recombinant anti-intimin antibody obtained was able to recognize the conserved region of intimin (Int388-667) in its purified form and α intimin in a typical EPEC isolate, and was more efficient than the native monoclonal antibody.
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Chen, Jianqing. "Radiolabeling and biotinylation of internalizing monoclonal antibody chimeric BR96 potential use for extracorporeal immunoadsorption with enhanced tumor radioactivity retention of iodine, indium and rhenium /". Lund : Lund University, the Jubileum Institute, Dept. of Radiation Physics, 1997. http://catalog.hathitrust.org/api/volumes/oclc/39725797.html.
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Congy-Jolivet, Nicolas. "Rôle majeur du FcyRIIIa/CD16a parmi les récepteurs activateurs des cellules tueuses naturelles (cellules NK) : etude de son expression et des réponses fonctionnelles induites par son engagement". Thesis, Tours, 2009. http://www.theses.fr/2009TOUR3132.
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Les cellules NK sont capables d’ADCC (Antibody Dependent Cytotoxicity) suite à l’engagement durécepteur Fc!RIIIa/CD16a, et de fonctions effectrices directes antivirales et anti-tumorales: c’est la«cytotoxicité naturelle ». Ainsi activées elles peuvent également répondre en produisant des cytokines, commel’IFN-!. La dégranulation et la synthèse d’IFN-! par les cellules NK observées après engagement du récepteurCD16a, dont l’expression est indépendante du polymorphisme V158F, ont été largement supérieures à cellesobtenues avec les autres récepteurs activateurs. Son engagement par les AcMor thérapeutiques a produit desréponses fonctionnelles variables selon l’AcMor, et selon les donneurs de cellules. La perte d’expression duCD16a membranaire s’est révélé être un marqueur sensible de l’activation des cellules NK, même quand cedernier n’était pas engagé. Enfin, l’emploi de d’inhibiteur d’ADAM17 (TMI-2 et TIMP3) a permis d’observerle maintien de l’expression du CD16a après activation cellulaire sans augmenter les réponses fonctionnelles.Ce travail souligne la place centrale de l’engagement du CD16a dans l’activation NKNK cell can trigger ADCC (Antibody Dependent Cytotoxicity) through the engagement of theFc!RIIIa/CD16a receptor, and « Natural Cytotoxicity » after integration of cellular signals coming from theiractivating and inhibitory receptors. Moreover, activated NK cells produce cytokines such as IFN-!.Engagement by monoclonal antibodies (mAb) of CD16a was strongly more efficient than that of any otheractivating receptor to induce degranulation and IFN-! synthesis. Functional responses depend on thetherapeutic mAb used to engage CD16a and on the donor of NK cells. CD16a down-modulation was a verysensitive marker of NK cell activation, whatever the mean of activation. It was inhibited in the presence ofTMI-2 and TIMP3 (ADAM17 inhibitors), whereas CD16-dependent functional responses were not increased.This work highlighted the major role of the CD16a receptor in the activation of NK cells
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Sabbaghi, Mehrjardi Mohammad Ali. "Uncivering mechanisms of acquired resistance to trastuzumab-emtansine (T-DM1) in HER2 positive breast cancer". Doctoral thesis, Universitat de Barcelona, 2017. http://hdl.handle.net/10803/456988.
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Trastuzumab-emtansine (T-DM1) is an antibody-cytotoxic agent (DM1) conjugated drug. DM1 delivery by trastuzumab inside the HER2 positive cells affects microtubule polymerization, cell cycle arrest and finally cell death. Although T-DM1 is approved for the treatment of HER2 positive metastatic breast cancer patients, primary and acquired resistance towards this drug is still a main challenge. Looking for the mechanisms of resistance is necessary to improve patient selection and to develop novel treatment strategies.
Here, we focused on finding mechanisms of acquired resistance to T-DM1 in a panel of HER2 positive breast cancer cell lines (HCC1954, HCC1419 and SKBR3 parental vs. resistant cells) generated by an established protocol of T-DM1 exposure, increasing the concentration of T-DM1[1-4µg/mL], 3days on/3days off, for 54 days overall.
We generated acquired resistant cells with different level of resistance to T-DM1 evaluated by 3, 7 and 10 days proliferation assay, using automated cell counting in SKBR3, HCC1419 and HCC1954 parental and the acquired resistant cells. Analysis of T-DM1 effects on cell cycle showed a significant induction of G2/M arrest in the parental cells, while this effect was not observed in the resistant cells. Expression/activity analysis of cyclin B1/CDK1 complex, the main apparatus involve in G2/M cell cycle arrest induction, showed a remarkable decrease in the basal level of cyclin B1 in the resistant cells. Cyclin B1 accumulation induced by T-DM1 in the parental cells was not observed in the resistant cells. CDK1 activity assay was also correlated with cyclin B1 expression, increasing following T-DM1 treatment in the parental cells, but not in the resistant cells.
Functional analysis revealed that cyclin B1 knock down in the parental cells induced a significant T-DM1 resistance. Furthermore, the silencing of cdc20, a protein mainly involved in APC complex related cyclin B1 degradation, could sensitize the resistant cells to T-DM1. Finally, cyclin B1 induction by T-DM1 was confirmed in in vivo and ex vivo xenograft animal model and patients’ explants, respectively. By cyclin B1 induction pattern, we could categorize T-DM1 responsive/non-responsive in fresh breast cancer explants from HER2 positive breast cancer patients.
Our results showed that T-DM1 induced G2/M cell cycle arrest in a cyclin B1/CDK1 dependent-manner. Lack of these effects appeared in acquired T-DM1 resistant cells. Besides, similar pattern in G2/M and cyclin B1 was verified in vivo and in patients explants. These data strongly suggest that induction of cyclin B1 is necessary for T-DM1 antitumor effects and emerges as a potential pharmacodynamic marker. Our finding also raises the question on what are the mechanisms leading to cyclin B1 dysregulation in resistant cells.
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Brunel, Simon. "Immunothérapie du cancer par administration d’anticorps monoclonaux anti-HVEM ou anti-ICOS chez la souris humanisée : potentiel thérapeutique et effets immunologiques". Thesis, Paris 6, 2017. http://www.theses.fr/2017PA066215.
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Un nouveau couple de récepteurs de co-signalisation (BTLA/HVEM) a récemment été proposé comme un acteur important dans l’échappement tumoral. L'expression d’HVEM a été identifiée dans la plupart des cancers. Son niveau expression est inversement corrélé avec la survie des patients. L'expression d’HVEM par des cellules tumorales pourrait inhiber la réponse immunitaire à travers BTLA exprimé par les lymphocytes T.Ainsi, dans notre travail, des anticorps monoclonaux (mAb) ciblant HVEM ont été évalués dans un modèle de souris NSG greffées avec des lymphocytes T et des tumeurs humaines. Nous avons tout d’abord caractérisé un clone anti-HVEM avec une forte affinité in vitro. Le clone choisi pour notre étude a montré sa capacité à favoriser l’activation des lymphocytes T humains in vivo, attesté par une aggravation des symptômes et de la mortalité associée au transfert de PBMC humains chez la souris. L’effet anti tumoral observé en l’absence de transfert adoptif était renforcé en présence de lymphocytes T, suggérant un effet additif de l’anticorps sur la tumeur et les lymphocytes T. Une diminution des lymphocytes T régulateurs et une augmentation de la prolifération des lymphocytes T CD8+ dans la tumeur était parfois associée à ce retard de croissance.En reproduisant un environnement partiellement humain chez la souris NSG, nous avons pu évaluer l'effet thérapeutique d’un mAb anti-HVEM dans le développement de deux types de tumeurs humaines et son impact sur le système immunitaire humain. Nos résultats indiquent qu’HVEM, de par son expression par la tumeur et les lymphocytes T, pourrait être une piste judicieuse pour l’immunothérapie du cancerA new pair of co-signaling receptors (BTLA / HVEM) has recently been proposed as an important actor in tumor escapement. HVEM expression has been identified in wild type of cancers. Its expression level is inversely correlated with patient survival. Expression of HVEM by tumor cells could inhibit the immune response through BTLA expressed by T lymphocytes.Thus, in our work, monoclonal antibodies (mAb) targeting HVEM were evaluated in a model of NSG mice grafted with human T cells and tumors. We first characterized an anti-HVEM clone with high affinity in vitro. The clone selected for our study showed its ability to promote activation of human T lymphocytes in vivo as evidenced by worsening symptoms and mortality associated with human PBMC transfer in mice. The anti-tumor effect observed in the absence of adoptive transfer was enhanced in the presence of T lymphocytes, suggesting an additive effect of the antibody on the tumor and T lymphocytes. A decrease in regulatory T lymphocytes and an increase in the proliferation of CD8 + T lymphocytes in the tumor was sometimes associated with this growth retardation.By reproducing a partially human environment in NSG mice, we were able to evaluate the therapeutic effect of an anti-HVEM mAb in the development of two types of human tumors and its impact on the human immune system. Our results indicate that HVEM, by its expression by the tumor and the T lymphocytes, could be a judicious track for the immunotherapy of the cancer
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Santos, Ana Paula Carneiro dos. "Construção e seleção de uma biblioteca de anticorpos monoclonais scFv contra celulas tumorais de tireoide". [s.n.], 2010. http://repositorio.unicamp.br/jspui/handle/REPOSIP/330245.
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Orientador: Laura Sterian WardDissertação (mestrado) - Universidade Estadual de Campinas. Faculdade de Ciencias Medicas
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Resumo: Fragmentos de anticorpos recombinantes têm se tornado ferramentas importantes em diversas áreas, tais como: Biologia Molecular, Farmacêutica e pesquisa Médica. Avanços recentes estão relacionados à aplicação desses anticorpos na oncologia, com estratégias diagnósticas e terapêuticas para diferentes carcinomas. Neste estudo, uma biblioteca de fragmentos de anticorpos monoclonais scFv foi construída utilizando RNA total de sangue periférico de 25 pacientes com Carcinoma Diferenciado da Tireóide. Essa biblioteca scFv foi selecionada utilizando os métodos Bioppaning and Rapid Analysis of Selective Interactive Ligands (BRASIL) e Phage display contra células tumorais de tireóide, com o objetivo de encontrar ligantes específicos a superfície celular tumoral. Os clones selecionados foram identificados por Dot blotting, e a reatividade contra proteínas de tumor, adenoma e bócio foi analisada por Elisa. O clone scFv-C1 apresentou melhor reatividade pelas proteínas tumorais e foi escolhido para a imunoistoquímica. Esta foi realizada com lâmina de Micro-arranjo de tecido (TMA) com duzentos e vinte nove casos de tireóide, sendo 110 Carcinomas, 52 Adenomas Foliculares, 49 Bócios e 18 tecidos normais de tireóide. O anticorpo scFv-C1 reagiu especificamente aos tecidos de câncer, com reatividade ao citoplasma das células tumorais, foi capaz de distinguir o Grupo Câncer do Controle (Bócio, Adenoma e tireóide normal) com significância estatística (p<0,0001) e entre os carcinomas reagiu melhor com os tumores pequenos (TNM 1 e 2) e com pouca agressividade (p=0,050). O fragmento de anticorpo scFv-C1 pode ser um potencial candidato a biomarcador para o diagnóstico do Câncer de tireóide
Abstract: Recombinant antibody fragments have become important tools in several fields, including molecular biology, pharmaceutical and medical research. In this study, a human single-chain variable fragment (scFv) antibody library was constructed using total RNA of leukocyte cells obtained from blood of patients with well differentiated thyroid carcinoma. This scFv antibody library was selected using the Biopanning and Rapid Analysis of Selective Interactive Ligands method (BRASIL) and Phage display technology against tumor thyroid cells, aiming to find specific cell-surface binders. The selected clones were identified by dot blot and ELISA assays and their reactivity analyzed against tumor, goiter and adenoma proteins. One clone (scFv-C1) presented the highest reactivity ratio between cancer and the control group (goiter and adenoma) and was chosen for further analysis. Immunohistochemistry was performed by means of Tissue Microarray with two hundred and twenty-nine thyroid cases (110 carcinomas, 52 follicular adenomas, 49 goiters and 18 normal tissues) including 38 papillary, 42 follicular and 30 variant follicular in the carcinoma group. The scFv-C1 reacted specifically to cancer tissues sections, showed strong reactivity with cytoplasm and was able to distinguish cancer to control groups (goiter, adenoma and normal thyroid) with_statistically significance (p<0,0001). The scFv-C1 fragment antibody described here may be a potential biomarker candidate for diagnostics and prognostics of thyroid cancer
Mestrado
Ciencias Basicas
Mestre em Clinica Medica