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1
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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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.
3
Duguid, I. G. M. "Prevention of corneal graft rejection with monoclonal antibodies." Thesis, University of Aberdeen, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.387460.
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This thesis aims to place corneal allograft rejection in the context of general transplantation immunology, examine the role of lymphocyte subsets in the rejection process and consider the potential application of monoclonal antibody therapy in clinical corneal graft rejection. The literature relating to the current clinical practice of corneal grafting, with particular reference to corneal allograft rejection, is reviewed in chapter 1 to present the extent of the problem. Chapter 2 then reviews the mechanisms of allograft rejection from the literature of transplantation immunology, much of which has arisen from studies of kidney, heart, pancreatic islets and liver in animal models. The materials and methods are described in detail in chapter 3, and only the relevant experimental design is detailed in the Materials and Methods sections of the succeeding chapters. The experimental mouse model of transplanting corneal tissue into the renal subcapsular is evaluated in chapter 4, demonstrating that isografts survive indefinitely whereas allografts are rejected typically by 30 days. Pretransplant sensitisation decreased allograft survival time to 10 days. Immunohistochemistry demonstrated the presence of CD4+ and CD8+ lymphocytes and macrophages at the rejection site. Heterotopic corneal graft recipients were then treated with various monoclonal antibody regimes. Chapter 5 demonstrates that allograft survival can be increased by either anti-CD4 or anti-CD8 therapy, providing near total depletion of the respective lymphocyte subset is achieved. Xenograft rejection is shown to depend on mainly CD4+ lymphocytes in chapter 6, with no benefit being found of depleting the CD8+ subset in addition. A mild immunosuppressive effect of anti-Vβ8 monoclonal antibody is demonstrated and discussed in chapter 7. The final chapter discusses these results in the light of recent, related work in other transplant systems, and presents a case for a trial of intracameral pan-T-cell monoclonal antibody treatment.
4
Abedian, Azadeh. "Definition of monoclonal antibodies specific for natural suppressor cells." Thesis, McGill University, 1990. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=59562.
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This investigation was carried out in order to determine the specificity of a panel of monoclonal antibodies previously generated against murine pregnancy-associated splenic natural suppressor cells and to characterize the cell surface target molecule(s) on cells that are recognized by these antibodies. The monoclonal antibody from clone 2C1.1 was purified on anti-rat Ig affinity column and used to partially characterize the antigenic determinant recognized by it. A 50 KDa target molecule was detected on pregnancy spleen cells enriched for Natural Suppressor activity but not on similar cells from the virgin animals. Experiments employing a cellular ELISA technique demonstrated that the antibody bound specifically to NS cells. It is proposed that the naturally occurring pregnancy-associated suppressor cells as defined by our specific anti-NS monoclonal antibodies may have physiological relevance in maintaining a state of immunological equilibration between the maternal and fetal environments by non-specifically downregulating potentially deleterious maternal anti-fetal immune reactions.
5
Lunn, David Paul. "Monoclonal antibodies recognising equine leucocyte surface antigens and immunoglobins." Thesis, University of Cambridge, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.387106.
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Durko, Margaret. "Identification of human organ-specific cancer neoantigen by monoclonal antibodies." Thesis, McGill University, 1986. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=75432.
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Soluble lung tumor antigen activity, as determined by the leukocyte adherence inhibition (LAI) assay, was enriched by physicochemical methods from chemically-defined spent medium of a lung cancer cell line (NCI-H69). To identify the polypeptide carrying the antigenic determinant, BALB/c mice were immunized with the enriched isolate and their splenic lymphocytes were fused with mouse plasmacytoma cells.Eight hybrids were cloned and produced MAbs that immunoprecipitated principally a single chain of MW 40,000 (p40) as well as minor chains of MW 25,000 (p25) and MW 13,000 (p13) which were probably degradation products of p40. On 2D gels, p40 was composed of 7 spots with a pI of 6.1 to 7.6, which was not altered by neuraminidase digestion. Affinity chromatography with MAb anti-p40 absorbed p40 and LAI activity. The bound and recovered fraction was enriched for p40 and LAI activity. Affinity-purified p40 contained the previously identified p25 and p13 as well as a MW 32,000 polypeptide (p32). MAb anti-p40 was directed to a common framework determinant on p40 since MAb anti-p40 bound to cancer cells from other organs. The lung cancer organ-specific determinant recognized by leukocytes from lung cancer patients was not recognized by the MAb. Affinity purified p40 triggered the LAI response for leukocytes from patients with lung cancer but not for leukocytes from control subjects or patients with colon cancer or malignant melanoma in blind testing. Partial cross-reactivity was observed with leukocytes from patients with breast cancer. Thus, a p40 molecule has been purified that is expressed on the membranes of lung cancer cells and triggers immunologically-mediated LAI.
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Dan, Michael. "Human anti-glioma monoclonal antibodies from patients with neurological tumors." Thesis, McGill University, 1989. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=74367.
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The current management of malignant gliomas is unsatisfactory compared to other solid tumors. Expected median survival is less than one year with even the best of care. At some point in their illness, most patients with neurological tumors are capable of mounting an immune response to their disease. This study focused on the humoral immune response of brain tumor patients by preparing human-human B cell hybridomas from autologous peripheral blood lymphocytes and a human myeloma-like cell line, designated as TM-H2-SP2. Eighteen fusions were successfully performed, and 15.8% of all microwells screened contained human immunoglobulin with anti-tumor activity. Five hybridomas, designated as BT27/1A2, BT27/2A3, BT32/A6, BT34/A5, and BT54/B8 were selected for detailed study. All five produced monoclonal IgM in a range of 2.4-44 $ mu$g/ml, had a similar (but not identical) pattern of reactivity against a panel of human tumor cell lines, and did not react with normal human astrocytes. All five human monoclonal antibodies (HmAbs) recognized a subpopulation of tumor cells based on multiparameter flow cytometric studies. Cell sorting experiments suggested that the identified subpopulation may share certain properties with hypothetical tumor stem cells. Preliminary antigen characterization indicated that the HmAbs are directed to cell surface glycolipids. These HmAbs possess certain properties of reactivity that suggest potential roles for them in the future diagnosis and clinical management of human malignant gliomas.
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Bolt, Sarah Louise. "Serotherapy with monoclonal antibodies directed to the human CD3 antigen." Thesis, University of Cambridge, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.240125.
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Smith, Richard. "Class II MHC specific monoclonal antibodies as immunosuppressive agents." Thesis, University of Cambridge, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.362802.
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Wiley, James A. "Monoclonal anti-idiotypes induce neutralizing antibodies to enterovirus-70 conformational epitopes." Thesis, University of Ottawa (Canada), 1992. http://hdl.handle.net/10393/7649.
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The model pathogen used in the development of the anti-idiotypic antibodies produced in this project was enterovirus-70. This virus is the causative agent of acute hemorrhagic conjunctivitis. In the past twenty-five years, this virus has been responsible for two worldwide pandemics of acute hemorrhagic conjunctivitis. Monoclonal antibodies (MAbs), directed against the prototype enterovirus-70 strain, J670/71, were generated and characterized in order to produce a monoclonal anti-idiotypic antibodies (MAb2s) for use as surrogate immunogens. Radio-immunoprecipitation and western immunoblot assays suggested that all the monoclonal antibodies recognize conformational epitopes on the virion surface. A neutralizing monoclonal antibody, MAb/ev-12, was selected for the production of MAb2s. Five MAb2s were selected for their capacity to inhibit the interaction of MAb/ev-12 with EV-70 in dot immunobinding inhibition and immunofluorescence assays. These five MAb2s also inhibited virus neutralization mediated by MAb/ev-12 suggesting that each recognizes a paratope associated idiotope. In competition enzyme immunosorbent assays, none of the five MAb2s recognized other neutralizing and non-neutralizing enterovirus-70 specific MAbs, thus demonstrating that the MAb2s were specific for private idiotopes. Immunization with each of the MAb2s was carried out for the production of anti-anti-idiotypic antibodies (Ab3). All five MAb2s induced an immune response. Moreover, results suggested that they share idiotopes since MAb2:MAb/ev-12 binding could be inhibited by homologous as well as heterologous Ab3. Ab3 sera were shown to possess antibodies capable of immunoprecipitating $\sp{35}$S-labelled viral proteins in the same manner as MAb/ev-12. Nine of fifteen mice immunized with MAb2s demonstrated Ab3 neutralizing activity specific for the prototype EV-70 strain, J670/71. The potential application of MAb2s to serve as surrogate immunogens for conformational epitopes is substantiated by the results presented in this report.
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MacIntyre, Elizabeth A. "Activation of human haemopoietic cells via Fc receptors for IgG." Thesis, University of Newcastle Upon Tyne, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.241359.
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Bailey, Graham D. "Generation of human monoclonal antibodies : the development of methods to identify and isolate neutralising antibodies targeted to Ebolavirus from peripheral blood." Thesis, University of Nottingham, 2017. http://eprints.nottingham.ac.uk/39911/.
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Neutralising antibodies are an essential component in the immune response to virus infection and are often effective at preventing or attenuating viral diseases. The administration of cocktails of potently neutralising monoclonal antibodies (nAb) to abrogate viral infections has been demonstrated as an effective treatment to control disease progression caused by a diverse range of viruses. The mechanism of neutralisation is mediated predominantly by nAb binding to glycosylated envelope proteins displayed on the surface of virus particles, thereby preventing binding to host cell membrane bound receptor proteins and neutralising the entry process. Although a robust nAb response is elicited following infection with a number of viruses, many human viruses have evolved mechanisms to effectively evade the humoral immune response. The emergence or re-emergence of pathogenic human viruses poses a significant threat to the global population. The Ebolavirus epidemic of 2014-2015 arose from the emergence of a known virus in a new area, which rapidly spread through a naïve population with devastating consequences, resulting in 11,323 fatalities from a total of 28,646 confirmed cases of infection. A rapid induction of potent, broadly nAbs was a possible pathway for infection control in the 17,323 survivors of the epidemic. The isolation of potent nAbs from seroconverted individuals following antigenic exposure provides the potential to develop novel therapeutics for administration within a region affected by an epidemic. To this end, the aim of this project was to develop a platform of methods to isolate human monoclonal antibodies directly from peripheral blood through the isolation of memory B-lymphocytes and subsequent stimulation to secrete antibodies using an in vitro culture system. Identification of DNA sequences encoding antibody heavy and light chain variable domains (VH and VL respectively), allowed for the production of recombinant antibody following the sub-cloning of VH and VL regions into plasmid expression vectors to provide a proof of principle of platform function. Secondly, the introduction of functional screens to the platform would provide a method to identify antibodies derived from memory B-cells with the ability to neutralise Ebolavirus pseudo-particles using in vitro entry inhibition assays.
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Dyrynda, E. A. "Studies on the immunology of the marine mussel Mytilus edulis L. using monoclonal antibodies." Thesis, Swansea University, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.636757.
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The initial aim of this project was to raise monoclonal antibodies to the haemocytes of the marine mussel, Mytilus edulis, which were capable of distinguishing subpopulations of haemocytes. Thirteen monoclonal antibodies were raised, including 3 antibodies for all haemocyte types, 2 specific for eosinophilic haemocytes and 8 specific for different subpopulations of basophilic haemocytes. All the antibodies were found to bind to the granules and/or cytoplasm of the respective haemocyte types. These monoclonal antibodies have illustrated some common links between phylogenetically related molluscs. A total of 11 other species were tested from 3 taxonomic orders. Cross-reactivity was most likely in those species closest taxonomically to M. edulis, and was also most probable with the non-selective antibodies. Functional studies have examined the effects of the monoclonal antibodies, and six others kindly provided by Dr. D. Noël (DRIM, University of Montpellier), on cell adhesion and phagocytosis. No significant differences were observed in phagocytosis, however, some indication exists of the involvement of monoclonal antibody 19E10 in cellular adhesion. The reactivity of the monoclonal antibodies with larval cells and adult tissue sections appears to be a promising technique for targeting potential haemopoietic sites. Reactivity with the antibodies was observed in undifferentiated larval cells and in adult connective tissue, the latter has previously been suggested as a haemopoietic site in bivalves. As part of the research into immune ontogeny, the immune response of the larvae of M. edulis has also begun to be characterized, using a disaggregation technique. To date, the enzymes arylsulphatase and phenoloxidase have been identified, and phagocytosis has been quantified in larval cells. The generation of superoxide anion has also been detected, although the levels observed were found to vary considerably with the techniques employed to manipulate the cells.
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Parsons, Stephen Frederick. "The use of monoclonal antibodies to study the components of the human erythrocyte membrane." Thesis, Open University, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.278501.
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Hoggatt, April Marie. "Mab anti-type I and Mab anti-zebrin II labelling in two siluriform fishes : the role of shared lineage versus shared function in polypeptide co-distributions." Virtual Press, 1994. http://liblink.bsu.edu/uhtbin/catkey/902481.
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Two monoclonal antibodies (mabs), the newly generated mab anti-type I and the previously documented mab anti-zebrin II, were reacted with brainstem sections of two ostariophysan siluriforms, the gymnotoid Rhamphichthys rostratus and the siluroid Ictalurus punctatus. Mab anti-type I recognizes a 47 kD polypeptide present in the dendrites and soma of projection neurons. Mab anti-zebrin II recognizes a 36 kD polypeptide present throughout the neuronal cytoplasm, including the axon. Strongly type I immunopositive cells include all cerebellar Purkinje cells, pyramidal cells of the nucleus medialis, electrosensory lateral line lobe, and tectum, pacemaker relay cells, Mauthner neurons, lateral line ganglion cells, and cells of the reticular formation, lateral reticular nucleus, and inferior olive. Weakly reactive type I cells include neurons in the torus semicircularis, medial and efferent octavolateralis nuclei, magnocellular and lateral tegmentum, and motor neurons of the Vth, V I Ith, and Xth cranial nerves. All type I positive cells are projection neurons. Zebrin II expression is restricted to subsets of two cell types which also express the type I antigen -- Purkinje cells and developing acousticolateralis pyramidal cells. Both of these neurons develop from the region of the rhombic lip. Thus, the mutual expression of the type I antigen can be explained by the shared function of projection neurons, while the common expression of the zebrin II antigen may be due to a shared embryological lineage.Department of Physiology and Health Science
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Peri, Delila. "Next generation monoclonal antibodies and their mechanisms of action against B-cell lymphomas." Thesis, University of Iowa, 2012. https://ir.uiowa.edu/etd/3367.
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Next generation monoclonal antibodies (mAbs) are unique in that they are specifically designed to enhance their mechanisms of action, primarily complement fixation and antibody-dependent cellular cytotoxicity (ADCC). Recent studies suggest that complement-fixing properties of a mAb can counter its ability to activate NK cells and mediate ADCC. GA101, a third generation (type II anti-CD20) mAb, and rituximab-MAGE (glyco-engineered type I mAb) show enhanced ADCC and direct cell killing; while ofatumumab, a second generation anti-CD20 mAb, shows enhanced complement-mediated cytotoxicity (CMC). These studies set out to determine the primary mechanisms of actions of these various mAbs, and compare the effect of complement on their ability to activate NK cells and mediate ADCC or CMC. We also studied the efficiency of rituximab vs. rituximab-MAGE to deplete B-cells in vivo in mice expressing human transgenic CD20. In vitro, rituximab and ofatumumab fixed more complement and mediated a greater degree of CMC, than GA101 and rituximab-MAGE. Additionally, complement inhibited the ability of both rituximab and ofatumumab to bind to and activate NK cells, whereas, addition of complement to GA101 or rituximab-MAGE did not affect their NK cell activating ability. Complement also blocked rituximab-induced NK-cell mediated ADCC, but not GA101-induced NK-cell mediated ADCC. Finally, GA101 and rituximab-MAGE depleted a higher percentage of B cells in whole blood compared to rituximab and ofatumumab, whereas rituximab-MAGE depleted fewer B cells, in vivo, in a complement-dependent fashion. We conclude from these studies that there are significant differences among these antibodies and that the ability of a given antibody to mediate CMC and complement fixation correlates with the ability of complement to block the interaction between the antibody and NK cells.
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Spencer, Valero Lilian Maritza. "Monoclonal antibodies binding to malarial merozoite surface protein 1 protect in vivo against plasmodium yoelii infection." Thesis, Open University, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.363486.
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Johansson, Fredrik. "Microscale measurement of kinetic binding properties of monoclonal antibodies in solution using Gyrolab." Thesis, Uppsala universitet, Institutionen för biologisk grundutbildning, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-155575.
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The number of monoclonal antibodies approved for therapeutic use has increased rapidlyover the last decade. As a consequence, precise and robust kinetic characterization techniquesare crucial in order to select the best suitable candidates. A kinetic characterization methodwas developed in Gyrolab with automated sample transfers. The characterization wasperformed in solution in a mixing CD, containing an integrated nanoliter mixing chamberwith affinity binding columns. Association rate constants were determined for four anti-TSHantibodies with values ranging from 3x105 M-1s-1 to 10x105 M-1s-1. The antibodies wereranked according to kass. Reproducibility
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Lodhia, Puja. "Investigating the intracellular interactions of CLEC14A and the characterisation of monoclonal antibodies targeting CLEC14A." Thesis, University of Birmingham, 2016. http://etheses.bham.ac.uk//id/eprint/7014/.
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CLEC14A is a tumour endothelial marker known to regulate sprouting angiogenesis. While the extracellular interactions of CLEC14A have previously been studied, the intracellular interactions of CLEC14A are unknown. Fascin was identified as a binding partner for the cytoplasmic tail of CLEC14A using a yeast two hybrid screen. Interaction of CLEC14A with fascin was confirmed by proximity ligation and co-localisation was observed in HUVEC filopodia. This data indicated that interaction of CLEC14A and fascin may be important for filopodia formation during sprouting angiogenesis. Binding studies with domain deletion mutants of fascin revealed the CLEC14A binding site to be located within a highly conserved region of the β-trefoil 3 domain between amino acids 323 and 384. In addition, phosphorylation of S274 was found to regulate this interaction. Five monoclonal antibodies against CLEC14A had the potential to be developed into anti-angiogenic cancer therapeutics. The functional properties of these antibodies were explored in in vitro assays. Clones 1 and 3 were found to inhibit cell migration while clone 4 disrupted tubule formation. Clones 3 and 4 were developed into antibody drug conjugates (ADCs). These ADCs demonstrated potent cytotoxicity localised to the tumour endothelium in vivo. These results indicate that targeting CLEC14A could be an effective strategy to disrupt the tumour vasculature.
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Wee, Christine. "Characterization of monoclonal antibodies against the T-cell protein tyrosine phosphatase and functional analyses of its promoter." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape7/PQDD_0016/MQ50901.pdf.
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Beckwith, Kyle Addison. "Novel Immunotherapeutic Strategies for Chronic Lymphocytic Leukemia." The Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1461203257.
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Clayton, Lisa Victoria Jane Eynstone. "Generation and characterisation of anti-C6 monoclonal antibodies in C6-deficient mice : the search for an anti-C6 therapy." Thesis, Cardiff University, 2006. http://orca.cf.ac.uk/54080/.
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For the second approach, monoclonal antibodies were raised against rabbit, rat, mouse and human C6. The two most interesting antibodies were raised against human C6 and inhibited complement-mediated haemolysis in a cell-based assay. Both of these antibodies were species specific, excluding the possibility of testing their therapeutic properties in animal models of complement-mediated disease. Instead, an ex vivo model of cardiopulmonary bypass was established and used to test the ability of these antibodies to block soluble C5b-9 formation. Neither antibody inhibited soluble C5b-9 formation, suggesting that they might be interfering with the insertion of C6 into the cell membrane during MAC assembly.
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Ogunwumi, Olumide Babatope. "The Development of Monoclonal Antibodies Against Human Immunodeficiency Virus-1 Viral Protein R Using Hybridoma Technology." Youngstown State University / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=ysu1441377756.
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Alkurbi, Mohammad. "Development and characterisation of anti-DBLβ surface-labelling and cytoadhesion-inhibitory mouse monoclonal and polyclonal antibodies." Thesis, University of Liverpool, 2016. http://livrepository.liverpool.ac.uk/2018781/.
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Plasmodium falciparum is responsible for most malaria-related morbidity and mortality, mostly affecting young children, non-immune adults and pregnant women. A characteristic feature of the pathogenesis of infection caused by P. falciparum is the cytoadherence of infected erythrocytes to the endothelial cells lining the microvessels of host organs. This phenomenon, termed ''sequestration'', mainly results from the adhesive interactions between P. falciparum erythrocyte membrane protein-1 (PfEMP1) proteins on the surface of infected erythrocytes and various host endothelial receptors such intercellular adhesion molecule 1 (ICAM-1), which is hypothesised to have a role in cerebral malaria. PfEMP1 molecules consist of several Duffy binding-like (DBL) and cysteine rich interdomain region (CIDR) domains that have different cytoadhesive functions. The second class of DBL domains, DBLβ, has been associated with adhesion to ICAM-1 receptors. In the present study, we selected four recombinant PfEMP1ICAM-1-DBLβ domains for mouse immunisations. Thirteen monoclonal (mAbs) and polyclonal antibodies (pAbs) were raised to three recombinant domains (DBL13, DBL31 and DBL41). All mouse mAbs and pAbs comprised IgM antibodies that recognised homologous and heterologous DBLβ domains. Most mAbs and pAbs labelled the surface of erythrocytes infected by P. falciparum isolates, with an IgM labelling capacity ranging from 10.1% to 67.6% of total IEs. Mouse antibodies showed similar patterns of reactivity with ICAM-1-binding and non-binding isolates, and reacted with a parasite isolate from a different genome (3D7). Surprisingly, we detected a remarkable reduction in IE population after incubation with mouse mAbs and pAbs, and this was mainly observed with antibodies that strongly labelled the surface of IEs. We demonstrated that this haemolysis was resulted from an immunological interaction between mouse IgMs and a parasite-derived component on the surface of live IEs. Antibodies raised to DBL41 were the most effective in all assays. Of these, three antibodies (pAb B5, mAb B4, mAb G6) and an anti-DBL31 mAb (E7) significantly blocked IE adhesion to purified proteins (ICAM-1 and CD36) under static and flow conditions. These antibodies also blocked parasite adhesion to HUVEC under conditions of blood flow. In a separate work, we characterised the immune response of eight semi-immune serum samples obtained from female adults living in Kilifi County, Kenya. Our results indicated that semi-immune sera specifically recognised five recombinant DBLβICAM-1 domains and a VAR2CSA DBL domain, and recognised the surface of erythrocytes infected by diverse parasite isolates with variable levels of reactivity. Some sera, particularly JA225 and JA235, significantly inhibited IE adhesion to ICAM-1 under both static and flow conditions. To our knowledge, this is the first study to examine the use of PfEMP1ICAM-1-DBLβ domains for the development of mouse mAbs and pAbs that recognise homologous and heterologous parasite isolates and block IE adhesion. However, further work is required to identify the surface ligand(s) involved in interaction with mouse IgM and to investigate the mechanisms of IgM-mediated IE lysis.
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Laffon, Leal Sandra M. "Development and screening of a marker to detect activated rainbow trout leukocytes." Thesis, University of Stirling, 2010. http://hdl.handle.net/1893/3025.
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Monoclonal antibodies (mAbs) have been essential tools in the elucidation of the immune system of mammals, and their application to identify surface molecules on leukocytes have allowed important functions of these cell to be identified (such as receptors that bind antigens, ligands involved in cell to cell signaling or in initiating immune response activity). Not only have mAbs been used to discriminate cells during different stages of cell development, but have also assisted in understanding the dynamics of molecules expressed during functional processes. Such molecules detected on human leukocytes are called human leukocyte differentiation antigens or HLDA. In order to group the antibodies that detect similar molecules and have similar patterns of reaction, immunologists have organised the mAbs that bind to these antigens into Clusters of Differentiation (CD). So far, there are about 350 leukocyte surface molecules detected by mAbs with a CD nomenclature for human leukocytes (www.hcdm.org). In fish immunology there is a great need to produce mAbs that are able to differentiate the various components of the fish immune system to assist in the elucidation of the fish immune system. The present study was an endeavour to develop and characterise mAbs that could be accredited to such scheme. A better understanding of the fish immune system is urgently required so that effective strategies of control can be developed for significant diseases during fish farming. Monoclonal antibodies were prepared by immunizing mice with thymic leukocytes from rainbow trout. The leukocytes were activated with the lectin Concanavalin A to promote the activation and proliferation of the target T-cell population. The selection of clones producing antibodies during screening was performed on the basis of the response of the supernatant from hybridomas using three consecutive assays. First, selection was determined by the positive staining of cells from the thymus in a Dot blot assay. Secondary screening was performed by means of flow cytometry (FCM) and the criterion for selection was the preferential detection of leukocytes gated in the lymphocyte region. Finally, the positive supernatants from hybridomes were evaluated to determine their effectiveness in the detection of modifications in the labelled cells during a multiple way activation by detection of foreign histocompatibility complex enhanced with mitogens. Monoclonal antibody TcOm15 was selected from 564 hybridomas produced and then used to stain cells from various Rainbow Trout tissues. It was clear from FCM, microscopy and Western blot analysis that mAb TcOm15 not only reacted with thymic cells but also with cells from other tissues. Differential staining of cells with mAb TcOm15 was observed with 27.1 ±1.4 % of leukocytes from peripheral blood leukocytes (PBL) stained in comparison to 2.0 ±0.2 % from the thymus, 13.8 ±0.4 % from the spleen, and 5.6 ±0.6 % cells stained from head kidney. The labeled cells showed characteristics of lymphocytes and monocytes, presenting a distinctive staining in immunohistochemistry and confocal microscopy. Western blot analysis, using electrophoresed proteins under denaturing conditions with leukocytes from several different tissues, showed that mAb TcOm15 did not detect a single protein. At least three proteins appeared to be identified by the mAb at 105, 160 and 200 kDa. The proteins were identified as α Actinin-4, non-erythroid Spectrin αII chain or Ig-like protein and non-muscle Myosin (MYH10) by MALDI-TOF analysis. Three of these identities are for compositional molecules for the cytoskeleton of different types of cells, and one it is associated to immunoglobulin superfamily. The identification of these proteins by mAb TcOm15 suggests an ability of this mAb to detect a specific function, possibly related with the synchronicity of expression or interaction of cytoskeleton-membrane proteins forming a multiprotein complex. Another possibility is as a carrier role for a protein during interactions. Colocalization of the mAb with F actin from the cytoskeleton was also observed suggesting the possibility that mAb TcOm15 detects a specific site in a multi-protein complex from the cytoskeleton. The molecule detected showed down-regulation in a dose dependant way with Concanavalin A and the expression was almost lost following stimulation of cells with phorbol 12-myristate 13-acetate stimulation. Leukocytes from the PBL and thymus up-regulated the expression of the TcOm15 molecule under mitogenic conditions in vitro, and results from in vivo experiments suggested the possibility of up-regulation on thymic cells. In conclusion, the results obtained in the present study provide information on a potentially useful marker (mAb TcOm15) for a cytoskeleton-membrane antigen that is modulated during stimulation of teleost lymphocytes. Additionally, this may enable insights into the relationship between cytoskeletal proteins and membrane associated immunoglobulin. Future research is necessary in order to explain this relationship and to determine the functional participation of the TcOm15 molecule during the activation of rainbow trout cells.
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Santis, Laís Priscila de. "Análise proteômica na caracterização de anticorpos monoclonais dirigidos contra antígenos eritrocitários e leucocitários humanos." Botucatu, 2018. http://hdl.handle.net/11449/154883.
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Orientador: Andrei MorozResumo: As membranas de hemácias e leucócitos são compostas por centenas de antígenos que desempenham diversas funções relacionadas a homeostase, metabolismo celular e podem estar envolvidos em processos de rejeição de transplantes, doenças hemolíticas e reações transfusionais. Para a detecção desses antígenos são utilizados anticorpos monoclonais e a obtenção destes anticorpos envolve diversas etapas que culminam na caracterização dos produtos obtidos. Esta etapa é crítica e envolve diferentes técnicas, incluindo a Proteômica na descrição da proteína-alvo de cada anticorpo monoclonal. O objetivo deste estudo foi caracterizar anticorpos monoclonais de especificidade anti-eritrocitária e anti-leucocitária produzidos pelo Laboratório de Engenharia Celular (LEC) do Hemocentro de Botucatu. Foram selecionados um clone e um hibridoma produtores de anticorpos anti-eritrocitários, e um clone produtor de anticorpos anti-leucocitários, pertencentes ao banco de células do LEC. As células foram expandidas em cultura, foi realizado Western Blotting (WB) e cada banda proteica reconhecida pelos anticorpos (antígenos) foi analisada por Espectrometria de Massas, segundo técnicas proteômicas. Outros testes adicionais foram realizados, como técnicas imuno-hematológicas, citometria de fluxo e imuno-histoquímica. Após expansão, retestagem e verificação de reatividade contra hemácias humanas, e a seleção dos dados de outros estudos até então não explorados, na técnica de WB os anticorpos reconheceram dive... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Red blood cell and leukocyte membranes are composed of hundreds of antigens that perform various functions related to homeostasis, cell metabolism and may be involved in transplant rejection, hemolytic disease and transfusion reactions. Monoclonal antibodies are used to detect these antigens and the obtaining of these antibodies involves several steps that culminate in the characterization of the obtained products. This step is critical and involves different techniques, including Proteomics to descript the target protein of each monoclonal antibody. The aim of this study was to characterize anti-erythrocyte and anti-leukocyte monoclonal antibodies produced by the Laboratory of Cellular Engineering (LEC) of the Blood Center of Botucatu. Anti-erythrocytes clone and hybridoma antibody producers and a clone that produces anti-leukocyte antibodies, belonging to the LEC cell bank were selected. Cells were expanded in culture, it was realyzed Western Blotting (WB) technique and each protein band recognized by the antibodies (antigens) was analyzed by Mass Spectrometry according to proteomic techniques. Others tests were realized, such as immunohematology techniques, flow cytometry and immunohistochemistry. After expansion, retesting and verification of reactivity against human red blood cells, and selection of data from other studies not exploited, in the WB technique, the antibodies recognized several spots. After analysis by Mass Spectrometry, it was identified, with good reliabi... (Complete abstract click electronic access below)
Mestre
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Rinehart, Erica Marie. "Inhibition of Nectin-1 and Herpes Virus Entry Mediator (HVEM) Using Monoclonal Antibodies Decreases HSV-1 Entry into Neuro-2A Cells." Wright State University / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=wright1438508507.
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Jarvis, Sandra Marie. "The application of immunology to food science, two studies : production of monoclonal antibodies (Mabs) specific for an enteropathogenic E. coli (EPEC) ; development of an enzyme-linked immunosorbent assay (ELISA) for [Beta]-N-acetylglucosaminidase (NAGase)." Thesis, University of British Columbia, 1989. http://hdl.handle.net/2429/28947.
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Two hybridoma clones, labelled 4D10 C1 and 2H4 H12, produced monoclonal antibodies which recognized the outer membrane of an enteropathogenic Escherichia coli (EPEC) 0142:K86:H6 in an enzyme-linked immunosorbent assay (ELISA) and the whole cell in an immunofluorescence assay. Large scale production of the monoclonal antibodies was accomplished through ascites production in balb/c mice. Purification of the ascites fluid was achieved by gel filtration and ion exchange chromatography. Isotyping of the purified fractions showed 4D10 C1 to be an IgG2 and 2H4 H12 an IgM. These monoclonal antibodies were screened by immunofluorescence assay against several pathogenic and nonpathogenic
strains of E.coli in addition to other Enterobacteriaciae. Results of the screening showed these antibodies to be specific for the E.coli serotype to which they were raised. Minimal cross-reactivity with other Enterobacteriaceae was observed.
In a separate and concurrent project, the use of an ELISA capable of detecting ß-N-acetylglucosaminidase (NAGase) was examined. White Leghorn hens were injected with commercially prepared bovine NAGase. Eggs were collected and the immunoglobulin fraction separated from the egg yolk by polyethylene glycol precipitation followed by ion exchange on a DEAE-Sephacel column. The use of the purified immunoglobulins was examined in a sandwich, double-sandwich and a competitive ELISA. A statistically significant standard curve for the detection of NAGase was successfully derived using a double-sandwich ELISA when rabbit immunoglobulin was used to coat the microwell plates. This assay was used to measure the NAGase concentration in press juice and fish extract of fresh and frozen salmon muscle samples. The ratio of the NAGase concentration in the press juice to the total NAGase concentration was compared. No significant difference was found between the calculated concentration ratios of the fresh muscle samples and samples frozen for 1 week at -20°C.Land and Food Systems, Faculty of
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Parthasarathy, Upasana. "Identifying epitopes of anti-FcaRI monoclonal antibodies on FcaRI ectodomain that trigger the anti-inflammatory ITAMi signaling pathway." University of Cincinnati / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1418910350.
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Baruah, Kavitha. "Structural biology of IgG Fc glycoforms." Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:def683d3-aa06-41d9-9f28-29d21258bebe.
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The conserved N-linked glycosylation site on the Fc domain of IgG1 antibodies is essential for maintaining a functionally active conformation of the antibody. Different glycoforms of the Fc exhibit widely different effector functions. Similarly, therapeutic antibodies, with engineered glycosylation, exhibit altered binding to cellular Fc receptors (FcRs). Here, X-ray crystallographic structures were obtained for biosynthetic intermediate glycoforms of human IgG1 Fc bearing: unprocessed oligomannose-type, intermediate hybrid-type, and mature complex-type glycans. The fully processed Fc protein crystallised in an “open” conformation with glycans forming canonical stabilising interactions on the protein surface. Analysis of the biosynthetic intermediates revealed that these stabilising hydrophobic protein-glycan interactions are formed only after processing by Golgi -mannosidase II. Mutagenesis of hydrophobic residues on Fc disrupted crucial protein-glycan interactions resulting in the selective destabilization of the 3-arm of the glycan chain with the 6-arm closely matching that seen for the native structure. However, carbohydrate analysis of released glycans shows increased processing on both arms indicating a more accessible and flexible glycan in the mutant structure suggesting that the crystallographic structure of these antibody glycans represents a minor low-energy conformation. The importance of Fc glycosylation is highlighted by endoglycosidases which eliminate Fc effector function. The crystallographic structure of enzymatically deglycosylated IgG Fc revealed a significant collapse of the of Cγ2 domains resulting in a ‘closed’ quaternary conformation, incompatible with Fc receptor binding. This provides a structural explanation for immune deactivating properties of endoglycosidases including those under preclinical development for the treatment of antibody-mediated immune pathology. One such bacterial endoglycosidase, Endo S, was studied further and revealed a specificity for complex-type glycans of the type found on IgG but no hydrolytic activity towards an engineered IgG Fc with oligomannose-type glycans. Introduction of both the engineered monoclonal IgG and endoglycosidase in serum led to a dramatic increase in FcR binding as the competitive binding of serum IgG for FcRs was selectively eliminated. This approach is a general technique for boosting the effector signal of therapeutic antibodies.
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Pullyblank, Anne Maria. "Evaluation of the role of monoclonal antibodies m17-1A, c17-1A and cSF25 in antibody-dependent cell-mediated cytotoxicity and an exploration of the possible mechanisms of action." Thesis, Imperial College London, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.268015.
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Barjon, Clément. "Caractérisation biochimique et fonctionnelle de nouveaux anticorps monoclonaux anti-galectine-9 en vue d'applications diagnostiques et thérapeutiques." Phd thesis, Université Paris Sud - Paris XI, 2013. http://tel.archives-ouvertes.fr/tel-00820984.
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La galectine-9 est une lectine animale principalement exprimée dans un contexte inflammatoire et possédant des propriétés à la fois pro-inflammatoires et immunosuppressives. Elle induit la production de cytokines inflammatoires par les cellules du système immunitaire inné tandis qu'elle induit l'apoptose des lymphocytes Th1 CD4+ et favorise l'expansion des lymphocytes Treg. Les propriétés immunomodulatrices de la galectine-9 dépendent en grande partie de son interaction avec le récepteur TIM-3. Cependant, ces deux molécules interagissent chacune avec d'autres protéines et dans plusieurs contextes la responsabilité de l'interaction galectine-9/TIM-3 n'a pas été formellement démontrée. Le développement d'un anticorps neutralisant les effets de la galectine-9 permettrait de préciser son rôle exact dans ces contextes. Par ailleurs, le blocage des voies de signalisations inhibitrices du système immunitaire est aujourd'hui un enjeu majeur en oncologie, comme le démontre le succès récent de la neutralisation du récepteur inhibiteur CTLA-4 pour le traitement des mélanomes. Nous avons produit de nouveaux anticorps monoclonaux dirigés contre la galectine 9 par immunisation de souris avec la partie C-terminale de la protéine. Parmi ces anticorps, 1G3 permet la détection de la galectine-9 sur coupes de tissus humains d'une manière très sensible et spécifique en immunohistochimie. Son utilisation nous a permis de confirmer l'expression constante et intense de la galectine-9 dans les cellules malignes de carcinome nasopharyngé et la forte expression de la galectine-9 dans les cellules de Kupffer présentes dans les tissus hépatiques infectés par le virus de l'hépatite C. Pour la première fois, nous avons mis en évidence une expression de la galectine-9 dans les leukocytes infiltrant les tissus hépatiques infectés par le virus de l'hépatite B. De plus, nous observons une expression de la galectine-9 dans les hépatocytes infectés par ces deux virus, ce qui n'avait pas été démontré jusqu'à présent. Nous avons également caractérisé les capacités fonctionnelles de nos anticorps lors de tests in vitro. L'anticorps 2E12 bloque la fixation de la galectine-9 au récepteur TIM-3 dans un test acellulaire, neutralise l'apoptose induite par la galectine-9 sur cellules de lymphomes T humaines et réduit considérablement l'augmentation de calcium cytosolique induite par la galectine-9 dans les cellules Jurkat. Ces effets de la galectine-9 sont indépendants de TIM-3 dans ce modèle cellulaire. L'anticorps 2E12 constitue un outil puissant pour étudier les fonctions de la galectine-9 à la fois dépendantes et indépendantes de TIM-3.
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Jansson, Eva. "Bacterial kidney disease in salmonid fish : development of methods to assess immune functions in salmonid fish during infection by Renibacterium salmoninarum /." Uppsala : Swedish Univ. of Agricultural Sciences (Sveriges lantbruksuniv.), 2002. http://epsilon.slu.se/avh/2002/91-576-6352-1.pdf.
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Czifra, György. "Identification of Mycoplasma gallisepticum antigens with diagnostic and protective properties /." Uppsala : Swedish Univ. of Agricultural Sciences (Sveriges lantbruksuniv.), 1999. http://epsilon.slu.se/avh/1999/91-576-5900-1.pdf.
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Garbergs, Hanna. "Extraction of therapeutic proteins from dried blood spots and their analysis on Gyrolab." Thesis, Uppsala universitet, Institutionen för biologisk grundutbildning, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-144920.
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A method for extraction of therapeutic proteins from dried blood spots (DBS) followed by quantification on Gyrolab(TM) has been developed. The method makes it possible to measure the concentration of the analyte in the range 100-6000 ng/mL. The procedure can generate full analytical information from 15 μL blood originally sampled from a subject. The modest sample requirements allows for sampling a full pre-clinical pharmacokinetic profile from a single mouse. This may allow for reduced usage of animals during preclinical development of new therapeutic proteins in accordance with the 3R’s, replace, refine and reduce.
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Costa, Matthew R. "FC Receptor-Mediated Activities of Env-Specific Monoclonal Antibodies Generated from Human Volunteers Receiving a DNA Prime-Protein Boost HIV Vaccine: A Dissertation." eScholarship@UMMS, 2010. http://escholarship.umassmed.edu/gsbs_diss/866.
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Human immunodeficiency type 1 (HIV-1) is able to elicit broadly potent neutralizing antibodies in a very small subset of individuals only after several years’ infection and as a result, vaccines that elicit these types of antibodies have been difficult to design. The RV144 trial showed that a moderate protection is possible, which may correlate with antibody dependent cellular cytotoxicity (ADCC) activity. Previous studies in the Lu lab demonstrated that in an HIV-1 vaccine phase I trial, DP6-001, a polyvalent Env DNA prime-protein boost formulation, could elicit potent and broadly reactive, gp120-specific antibodies with positive neutralization activities along with multiple Fc mediated effector functions. I developed a protocol for the production and analysis of HIV-1 Env-specific human monoclonal antibodies (mAbs) isolated from these DP6-001 vaccinees. By utilizing a labeled gp120 bait to isolate Env specific B cells, paired heavy and light chain immunoglobulin (Ig) genes were cloned and allowed for the production of monoclonal antibodies with specificity for gp120. By using this protocol, 13 isolated mAbs from four DP6-001 vaccinees showed broad binding activities to gp120 proteins of diverse subtypes, both autologous and heterologous to vaccine immunogens, with mostly conformational epitopes and a few V3 and C5 specific mAbs. Equally cross-reactive Fc-mediated functional activities, including ADCC and antibody dependent cellular phagocytosis (ADCP), were present with both immune sera and isolated mAbs, confirming the induction of non-neutralizing functional antibodies by the DNA prime- protein boost vaccination. Elicitation of broadly reactive mAbs by vaccination in healthy human volunteers confirms the value of the polyvalent formulation in this HIV-1 vaccine design.
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Costa, Matthew R. "FC Receptor-Mediated Activities of Env-Specific Monoclonal Antibodies Generated from Human Volunteers Receiving a DNA Prime-Protein Boost HIV Vaccine: A Dissertation." eScholarship@UMMS, 2016. https://escholarship.umassmed.edu/gsbs_diss/866.
Full textAbstract:
Human immunodeficiency type 1 (HIV-1) is able to elicit broadly potent neutralizing antibodies in a very small subset of individuals only after several years’ infection and as a result, vaccines that elicit these types of antibodies have been difficult to design. The RV144 trial showed that a moderate protection is possible, which may correlate with antibody dependent cellular cytotoxicity (ADCC) activity. Previous studies in the Lu lab demonstrated that in an HIV-1 vaccine phase I trial, DP6-001, a polyvalent Env DNA prime-protein boost formulation, could elicit potent and broadly reactive, gp120-specific antibodies with positive neutralization activities along with multiple Fc mediated effector functions. I developed a protocol for the production and analysis of HIV-1 Env-specific human monoclonal antibodies (mAbs) isolated from these DP6-001 vaccinees. By utilizing a labeled gp120 bait to isolate Env specific B cells, paired heavy and light chain immunoglobulin (Ig) genes were cloned and allowed for the production of monoclonal antibodies with specificity for gp120. By using this protocol, 13 isolated mAbs from four DP6-001 vaccinees showed broad binding activities to gp120 proteins of diverse subtypes, both autologous and heterologous to vaccine immunogens, with mostly conformational epitopes and a few V3 and C5 specific mAbs. Equally cross-reactive Fc-mediated functional activities, including ADCC and antibody dependent cellular phagocytosis (ADCP), were present with both immune sera and isolated mAbs, confirming the induction of non-neutralizing functional antibodies by the DNA prime- protein boost vaccination. Elicitation of broadly reactive mAbs by vaccination in healthy human volunteers confirms the value of the polyvalent formulation in this HIV-1 vaccine design.
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Gray, Daniel Herbert Donald. "Thymic stromal cells : population dynamics and their role in thymopoiesis." Monash University, Dept. of Pathology and Immunity, 2003. http://arrow.monash.edu.au/hdl/1959.1/9409.
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Silva, Fabiana Érica Vilanova da. "Reatividade específica e cruzada de antígenos de Echinococcus granulosus e Taenia crassiceps utilizando amostras de pacientes com hidatidose e neurocisticercose." Universidade de São Paulo, 2007. http://www.teses.usp.br/teses/disponiveis/9/9136/tde-11102017-181551/.
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Os estudos de reatividade dos antígenos de líquido hidático de Echinococcus granulosus (Ag LH-Eg) e de líquido vesicular de Taenia crassiceps (Ag LV-Tcra) foram feitos com anticorpos monoclonais (AcMos) anti-E. granulosus e anti-T. crassiceps e amostras humanas por eletroforese em gel de poliacrilamida contendo dodecil sulfato de sódio (SDS-PAGE), Enzyme-Linked Immunosorbent Assay (ELISA) e immunoblot. O SDS-PAGE mostrou um padrão complexo de proteínas entre 97- e 8-kDa do LH-Eg e 97- e 14-kDa do LV-Tcra. A caracterização dos antígenos com os AcMos por ELISA mostrou que todos os AcMoss anti-E . granulosus reagiram com o antígeno LH-Eg e um deles cruzada mente com o antígeno LV-Tcra. Um dos dois AcMo anti-T . crassiceps reagiu também com o LH-¬Eg. A reatividade do LH-Eg com amostras humanas apresentaram melhores resultados com o cut off média mais dois devios-padrão (M+2DP) e na diluição 1:250. As amostras de hidatidose (Hi) mostraram reatividade máxima (100%) nessa diluição. As amostras neurocisticercose (NC) ensaiadas por ELlSA-LH-Eg, apresentaram reatividade cruzada de 66,5%, teníase (T) 46%, controle negativo (CN) 4% e outras parasitoses (OP) 84,5%. O ELlSA-LV-Tcra mostrou 100% de reatividade com as amostras NC na diluição 1 :50 pelo cut off TgRoc. Com as amostras hidatidose, teníase, outras parasitoses e controle negativo a reatividade foi de 73,5; 61,5; 69 e 2%, respectivamente. Todos os AcMos anti-LH-Eg reconheceram as frações 79-,67- e 8-kDa do Ag LH-Eg e as de 24- e 16-kDa pelo AcMo anti¬-antígeno rAgB. No immunoblot, as amostras humanas reconheceram as frações 79-, 67-, 57-, 43-, 38-kDa e também as específicas (24-, 16-e 8-kDa) para o diagnóstico da hidatidose. As frações responsáveis pela reatividade cruzada foram 79-, 67-e 57¬kDa. Nossos estudos mostraram que é necessária uma melhor abordagem incluindo em estudos futuros a obtenção de antígenos recombinantes, específicos de cada um dos parasitos.Studies to evaluate the reactivity of the hydatic liquid antigens of Echinococcus granulosus (Ag LH-Eg) and of the vesicular liquid antigens of Taenia crassiceps (Ag LV-Tcra) were conducted using anti-E. granulosus and anti-T . crassiceps monoclonal antibodies (MoAbs), and human sample, through SDS-PAGE, ELISA and immunoblot tests. The SDS-PAGE showed a complex standard of proteins from 97¬to 8-kDa of the LH-Eg and from 97-to 14-kDa of the LV-Tcra. The characterization of antigens with the MoAbs through ELISA showed that ali the anti-E . granulosus MoAbs reacted with the LH-Eg antigen and one of them cross-reacted with the LV¬-Tcra antigen. One of the two anti-T. crassiceps MoAb also reacted with the LH-Eg antigen. The reactivity of the LH-Eg with human samples presented better results with the cut off value representing the mean value plus two standard deviations (M+2DP) and with a dilution of 1:250. The hydatidosis samples (Hi) showed maximum reactivity (100%) with this dilution. When evaluated by the ELlSA-LH-Eg, the neurocysticercosis samples (NC) showed cross-reactivity of 66,5%, the taeniasis (T) showed 46%, the negative control (CN) of 4% and 84,5% for other parasitoses (OP). The NC samples, diluted at 1:50, showed 100% of reactivity in ELlSA-LV-Tcra test with the TgRoc cut off value. Concerning the samples of hydatidosis, taeniasis, other parasitoses and the negative control, reactivity was 73,5; 61,5; 69 and 2%, respectively. Ali the anti-LH-Eg MoAbs recognized the fractions 79- and 67-kDa of the Ag LH-Eg and the 24-and 16-kDa through the rAgB anti-antigen MoAb. By immunoblot, the human samples recognized the fractions 79-, 67-, 57-, 43-, 38-kDa and also the specific ones (24-, 16-and 8-kDa) for diagnosing hydatidosis. The fractions, responsible for the cross-reactivity, were 79-, 67-and 57-kDa. Our study showed that further approaches are needed and should include the obtaining of recombinant antigens, specific for each parasite.
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Anthony, Robert McCullough. "Characterization and function of the inflammatory response to infection by a gastrointestinal nematode parasite : new insights into protective Th2 responses /." Download the dissertation in PDF, 2006. http://www.lrc.usuhs.mil/dissertations/pdf/Anthony2006.pdf.
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Gertner-Dardenne, Julie. "Optimisation de l'activité anti-tumorale des lymphocytes T gamma9delta 2 humains." Toulouse 3, 2008. http://thesesups.ups-tlse.fr/195/.
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Si tous les vertébrés possèdent des lymphocytes T gamma delta, seuls les primates ont une sous-population sanguine exprimant un récepteur de type TCR Vgamma9Vdelta2. Cette dernière réagit à des phosphoantigènes non-peptidiques d'origine bactérienne et tumorale, déclenchant ainsi son activité anti-infectieuse et anti-cancéreuse. Cette thèse traite de l'optimisation de son potentiel thérapeutique anti-cancéreux. Ces lymphocytes cytotoxiques capturent des fragments membranaires de leurs cellules cibles et sécrètent leurs granules cytolytiques, à travers leur synapse immunologique. L'étude par vidéo-microscopie de l'activité moléculaire à la synapse des lymphocytes T gamma delta révèle la simultanéité de ces deux processus. Ainsi, le renforcement de l'attachement des lymphocytes T gamma delta à leurs cibles par la combinaison de phosphoantigène et d'anticorps thérapeutiques permet d'optimiser leur efficacité anti-tumorale. Cette thèse présente donc les bases moléculaires d'une nouvelle approche thérapeutique anti-cancéreuseAlthough all vertebrates possess gamma delta T lymphocytes, only primates have a blood-borne gamma delta T cell subset expressing a TCR Vgamma9Vdelta2 receptor. This population reacts to bacterial and tumoral non-peptidic phosphoantigens, triggering its anti-infectious and anti-cancer activity. This thesis deals with the optimization of the therapeutic anti-cancer activity of TCR Vgamma9Vdelta2 gamma delta T lymphocytes. These cytotoxic effectors capture membrane fragments of target cells and release cytolytic granules through their immunological synapse. The timelapse video-microscopy study of the molecular activity at the gamma delta T lymphocytes synapse shows both processes simultaneously. So, the strengthening of the gamma delta T lymphocytes synapses to their target through a combination of phosphoantigen and therapeutic antibody strongly optimizes their anti-tumor efficacy. This thesis presents the molecular basis for a new anti-cancer therapeutic approach
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Hartmann, Lucie. "Développement et caractérisation d'outils immunologiques dirigés contre des récepteurs membranaires d'intérêt thérapeutique." Thesis, Strasbourg, 2019. http://www.theses.fr/2019STRAJ017/document.
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Les Récepteurs Couplés aux Protéines G (RCPG) constituent la plus grande famille de protéines membranaires chez l’Homme, et leur implication dans un grand nombre de processus physiologiques justifie pleinement l’intérêt de leur étude. Les anticorps spécifiques de ces récepteurs sont des outils polyvalents à haute valeur ajoutée, qui restent toutefois encore trop rarement disponibles, notamment en raison des difficultés techniques posées par leur génération. Ce manuscrit présente la mise au point d’une méthode d’immunisation alternative et innovante, mettant en jeu des particules virales recombinantes dérivées du Virus de la Forêt de Semliki (SFV) codant pour le récepteur d’intérêt. Appliquée au récepteur de l’adénosine A2A humain, l’immunisation permet d’engendrer la surexpression de celui-ci à la surface des cellules de l’animal infecté, et de provoquer l’apparition d’une réponse immunitaire. Cette approche permet d’une part de générer un sérum polyclonal de souris spécifique au récepteur, et ouvre donc une nouvelle voie pour l’obtention d’anticorps monoclonaux murins. Elle semble d’autre part prometteuse pour la génération de nanobodiesG Protein Coupled Receptors (GPCRs) constitute the largest membrane protein family represented in the human genome. Their involvement in a wide number of biological processes fully supports their study. GPCR-targeting antibodies are versatile and valuable tools, which remain scarcely available, chiefly because their generation is a challenging process. This thesis presents an alternative and innovative strategy in which recombinant Semliki Forest Virus (SFV) particles coding for the receptor of interest are used as immunogens. When applied to the human version of the Adenosine A2A receptor, this method enables to cause the receptor’s overexpression at the surface of the infected animal cells, which generates an immune response. This strategy enables to raise receptor-specific mouse polyclonal serum. It opens a new path towards the generation of monoclonal mouse antibodies. Additionally, it seems to also be a promising approach to develop nanobodies
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Edwards, Aaron David. "Production of functionality enhanced monoclonal antibodies via gene therapy." Thesis, 2014. https://hdl.handle.net/2144/15370.
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While the last century of medical discoveries has made a significant impact on improving the lives of human populations across the globe, a perfect solution to the yearly infection cycle from the influenza virus has yet to be discovered. Although vaccines stand the best chance at targeting yearly epidemics, new treatment options must be created to combat the arrival of rapidly mutating and antiviral-resistant strains of the virus that could lead to another pandemic such as the 1918 Spanish flu that killed millions worldwide. We describe a method to create functionally enhanced monoclonal antibodies targeting influenza via genetic engineering of fragment crystallizable glycan structures. Muscle and liver cell lines were lentivirally-transduced to produce the broadly neutralizing antibody, Fi6v3, while also overexpressing a critical glycosylation enzyme, B-1,4-N-acetyl-glucosaminyltransferase III. Secreted antibodies were tested for effector functionality using a Natural Killer cell degranulation assay and an antibody-dependent cellular phagocytosis assay. Results conclude that modified antibodies from both muscle and liver cells lines exhibit enhanced function in comparison to their unmodified counterparts, providing support to the future creation of an influenza prophylactic or treatment option using antibodies with the ability to more effectively activate innate immune killing mechanisms.
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Vendramelli, Robert Matthew. "Characterization of anti-ricin monoclonal antibodies and the construction of a chimeric murine-human IgG2/K anti-ricin monoclonal antibody." 2011. http://hdl.handle.net/1993/4526.
Full textAbstract:
Ricin toxin is a very deadly plant protein that is synthesized by the plant Ricinus communis. The molecular structure of ricin toxin places it in a group of similar proteins classified as a Type II RIP due to its heterodimeric construction; it is composed of a toxic A-chain possessing enzymatic action, and a receptor binding B-chain. Monoclonal antibodies were obtained with binding activities against either the A-chain or B-chain, and a surrogate non-toxic ricin analogue, TST10114, was determined to be suitable for characterization of the anti-ricin monoclonal antibodies. One potent anti-ricin A-chain neutralizing monoclonal antibody was chosen for chimerization, RAC18, which exhibited strong binding affinity and neutralizing properties. The constant regions of a human immunoglobulin G2 (IgG2) were used as the backbone for the recombinant chimeric antibody. The resulting chimeric RAC18-huG2 was transiently expressed in human-derived HEK 293F cells, purified, and assessed for binding characteristics and functional attributes.
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"Studies of oestrogen and progesterone receptors in human endometrium in menstrual cycle using monoclonal antibodies." Chinese University of Hong Kong, 1992. http://library.cuhk.edu.hk/record=b5887099.
Full textAbstract:
Wong Yuk-Ling.Thesis (M.Phil.)--Chinese University of Hong Kong, 1992.
Includes bibliographical references (leaves 132-152).
abstract --- p.1
ACKNOWLEDGEMENT --- p.4
content --- p.6
Chapter I. --- INTRODUCTION --- p.8
Chapter II. --- literature reviews --- p.11
Chapter 1. --- Menstrual cycle --- p.11
Chapter 2. --- oestrogen receptor and progesterone receptor --- p.18
Chapter 3. --- Monoclonal antibody assays for the study of oestrogen and progesterone receptors --- p.30
Chapter III. --- materials and methods --- p.35
Chapter 1. --- study population --- p.35
Chapter 2. --- sample collection and analysis JO --- p.36
Chapter 3. --- Histological dating of endometrial biopsies --- p.37
Chapter 4. --- Determination of oestrogen and progesterone receptors using immunocytochemical assay --- p.38
Chapter 5. --- Determination of oestrogen and progesterone receptors using enzyme immunoassay --- p.52
Chapter 6. --- Determination of serum oestradiol and progesterone --- p.66
Chapter 7. --- Data handling and statistical analysis --- p.76
Chapter IV. --- results --- p.77
Chapter 1. --- Study population --- p.77
Chapter 2. --- Histological dating of endometrial biopsies --- p.77
Chapter 3. --- oestrogen and progesterone receptors in frozen section of endometrium in menstrual cycle --- p.80
Chapter 4. --- oestrogen and progesterone receptors in paraffin section of endometrium in menstrual cycle --- p.95
Chapter 5. --- Oestrogen and progesterone receptors in endometrium in menstrual cycle determined by enzyme immunoassay --- p.113
Chapter 6. --- Serum oestradiol and progesterone --- p.116
Chapter V. --- DISCUSSIONS --- p.120
Chapter 1. --- oestrogen and progesterone receptors in frozen section of endometrium in menstrual cycle --- p.121
Chapter 2. --- Oestrogen and progesterone receptors in paraffin section of endometrium in menstrual cycle --- p.124
Chapter 3. --- oestrogen and progesterone receptors in endometrium in menstrual cycle determined by enzyme immunoassay --- p.127
Chapter 4. --- Potential application of oestrogen and progesterone receptors in endometrium in menstrual cycle --- p.129
REFERENCE --- p.132
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Shivakumar, Adarsha. "Antigens and cancer pathways targeted by de-N-acetyl polysialic acid monoclonal antibodies." Thesis, 2017. https://hdl.handle.net/2144/23840.
Full textAbstract:
Polysialic acid (PSA) is a developmentally regulated glycan made of repeating sialic acid monomers with α2-8 linkages. PSA has very limited expression in adults, and modifies only a few cell-surface proteins. However, PSA is overexpressed in several human cancers and is associated with metastasis and poor prognosis. We have described a derivative of PSA containing a mixture of de-N-acetyl and N-acetyl neuraminic acid residues (dPSA) found intracellularly in many normal human tissues but expressed at much higher levels on the cell surface of many human cancer cell lines. The proteins modified with dPSA and dPSA function in normal and abnormal human biology are unknown. The purpose of this study was to identify protein(s) modified with PSA and possible dPSA-dependent functions in cancer cell lines that express dPSA antigens. Using co-immunoprecipitation with the anti-dPSA monoclonal antibody SEAM 2 and mass spectroscopy, we identified membrane-associated nucleolin that is either directly modified or associated with dPSA. In addition, knocking down expression of the polysialyltransferase ST8SiaII (STX) in SK-MEL-28 human melanoma cells nearly eliminated dPSA and nucleolin from membranes but had no effect on the levels of nuclear nucleolin, and resulted in aberrant cell morphology, cell adhesion, and motility. The data suggest that cell-surface nucleolin depends on modification with dPSA, and that dPSA-modified nucleolin has an important role in cell adhesion and migration.
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Sarna, Caitlin S. "Cloning and characterization of novel IgA antibody variable heavy and light chains from HIV-1 resistant sex workers from Nairobi, Kenya." 2011. http://hdl.handle.net/1993/4589.
Full textAbstract:
Heterosexual intercourse now accounts for the majority of HIV transmission
within sub-Saharan Africa. The generation of microbicides and vaccines, therefore, requires a better understanding of the mucosal correlates of protection, including the role of HIV-specific IgA.
It is now accepted that not all individuals are equally susceptible to HIV-1 infection, as exemplified by the HIV Exposed Seronegative (HESN) women of the Pumwani Cohort in Nairobi, Kenya. To assess whether mucosal IgA responses contribute to this protection, 3 novel IgA variable genes were cloned from HESN cervical B-cell cDNA.
Nine monoclonal IgA Abs were produced, two of which were properly produced
from cell culture. The HESN-derived A6/30L and A9/30L variants had a greater specificity for gp120IIIB than their A6/4L and A9/4L counterparts, while the A6 variant recognizes a distinct gp120 epitope compared to the broadly neutralizing antibody IgGb12. Further characterization of these IgA chains may suggest their suitability for use in microbicides or mucosal vaccines.
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Gaudet, Ryan G. "The Isolation of gp41 Specific Monoclonal Antibodies from the Cervical IgA Repertoire of Highly Exposed Persistently Seronegative (HEPS) Commercial Sex Workers from Nairobi, Kenya using Mammalian Cell Display." 2010. http://hdl.handle.net/1993/3910.
Full textAbstract:
The mucosal antibody repertoire of the cervical mucosa in commercial sex workers from Nairobi, Kenya, who are highly sexually exposed to human immune deficiency virus type-1 (HIV-1) but remain persistently IgG seronegative (HEPS), may represent a novel source of broadly neutralizing monoclonal antibodies (mAbs) against HIV-1. Mucosal IgA specific for HIV-1 envelope (Env) subunit gp41 has been suggested as a correlate of protection in HEPS individuals. The in depth studies at both the gene and function level required to confirm their role in HIV-1 resistance are possible only using recombinant monoclonal IgAs. Human mAbs have traditionally been selected from libraries displayed on the surface of microorganisms (phage, yeast). However, due to inherent limitations, such techniques may not be optimal for isolating such rare mAbs from a pool of cervical B cells. We have developed an antibody selection system based on surface display on mammalian cells and used this technology to isolate four novel monoclonal antibodies, against linear epitopes on gp41, from the IgA repertoire of the cervical mucosa in Kenyan HEPS. Furthermore, three of the four mAbs were shown to bind with surface expressed consensus clade B and clade C Env on mammalian cells. Characterization of the variable region cDNA of the two strongest binding mAbs reveals extensive somatic mutations with a bias of replacement mutations clustering in the complementary determining regions (CDR) indicating antigen-driven affinity maturation had occurred. Affinity matured monoclonal IgAs, such as these, may play a role in the identification of new, vulnerable epitopes on HIV-1, or act as a component in a topical microbicide.
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Gubbala, Supreetha. "Characterization of Fc receptor family proteins in vaginal and endocervical epithelia." Thesis, 2014. https://hdl.handle.net/2144/14659.
Full textAbstract:
In the age of highly active antiretroviral therapy (HAART), patients infected with Human Immunodeficiency Virus Type 1 (HIV-1) are now living significantly healthier and longer lives. However, HIV prevention and cure still remain significant challenges. Globally, women face specific barriers to using and accessing both female and male condoms, the primary method recommended by the World Health Organization (WHO) to prevent sexual transmission of HIV-1. Although HAART treatment as prevention (TasP) of HIV has shown promising preliminary results, poor economic feasibility of the method in resource poor settings has yet to be resolved. Since women carry over 50% of the disease burden, there is a significant need for the development of a female-controlled method of prevention. One such approach is the reformulation of topical vaginal microbicides. Our laboratory is developing HIV-targeted microbicide formulations that utilize highly specific, broadly neutralizing anti-HIV antibodies (bNAbs).
The purpose of my research project was to characterize Fc receptor expression in epithelial cell models of the lower female genital tract with a particular focus on the neonatal Fc receptor (FcRn). Fc receptors are a large family of proteins that bind to the Fc region of immunoglobulins (Igs) and function in Ig transport and effector functions. These receptors could function in enhancing the delivery of bNAbs in microbicide formulations or potentially serve as a mechanism of delivering HIV to target cells in tissues via transport of HIV-antibody complexes. Thus, this thesis assesses Fc receptor expression in human vaginal and endocervical organotypic cultures via microarray, quantitative RT-PCR, immunohistology and preliminary functional assays.
Microarray results revealed significant expression of Fc receptor family genes in the epithelial cells of the lower female reproductive tract (FRT). The polymeric immunoglobulin receptor (pIgR), a well-characterized receptor that transports secretory IgA across mucosal epithelia, was abundantly expressed and hormonally regulated in epithelial cells of the vagina and endocervix. FcRn, a receptor originally characterized in the placenta and gut where it confers passive immunity from mother-to-child via bidirectional IgG transcytosis, was expressed in both tissue models. Moreover, several members of the novel FC receptor-like (FCRL) family were detected by microarray in both models.
Immunohistological staining revealed pIgR protein in the endocervical mucosal epithelium, confirming current literature describing its expression in the FRT and role in local production of cervicovaginal secretions. FcRn protein expression was detected in the basal cell layer of the stratified squamous vaginal epithelium and in the columnar cells of the endocervix. Preliminary functional assays did not observe FcRn-specific transcytosis of human IgG across vaginal or endocervical epithelia by ELISA or immunohistology. VRCO1, a monoclonal antibody in development for application in microbicide formulation, crossed the epithelium, but was likely not transcytosed via FcRn because immunohistology revealed the presence of antibody between epithelial cells rather than the expected intracellular localization of IgG utilized in the FcRn mechanism.
These preliminary findings indicate that Fc receptors, pIgR and Fc receptor-like proteins may play an important role in antibody-mediated immune responses in the FRT. Further research is require to determine whether FcRn functions in HIV-antibody complex-mediated HIV transmission or monoclonal antibody transcytosis.
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Lawson, Maxx. "Modulating the T cell response: using anti-interleukin-7 receptor-alpha monoclonal antibodies with autoantigen-specific immunotherapy to prevent type-1-diabetes." Thesis, 2019. https://hdl.handle.net/2144/37107.
Full textAbstract:
Autoimmunity develops over an extended period of time as the result of an amalgamation of genetic, environmental, and immunologic events. Though the precise etiological factors leading to most autoimmune disease are awaiting consensus, a common thread of the autoimmune paradigm is the inappropriate activation of tissue-specific immune cells by one or more autoantigen, which begins the destruction of the tissue. To prohibit immunopathology and fine-tune the immune responses in healthy individuals, the stimulatory activities of effector/memory T (Teffs) cells must be counteracted by the suppressive mechanisms of regulatory T cells (Tregs). Thus, the potential to modulate the ratio between Teff and Tregs in autoimmune patients has been widely investigated with high hopes to permanently cure certain autoimmune diseases such as type 1 diabetes militus (T1D). Autoantigen therapies, which attempt to induce Tregs to suppress pathogenic effector cells in an autoantigen-specific manner, have shown efficacy in preventing T1D in mice, but have largely failed in clinical trials. One approach to improve the effectiveness of islet autoantigen vaccinations is to combine them with an additional modulator of the T cell response which favors a regulatory phenotype. In the work presented here, we asked whether the addition of anti-interleukin-7 receptor-alpha (anti-IL-7Rα) monoclonal antibodies (mAbs) to islet autoantigen immunizations would modulate the T cell response and prevent T1D in non-obese diabetic (NOD) mice. It was found that anti-IL-7Rα mAbs reduced the absolute numbers of islet antigen-specific T cells when immunized with islet peptide in conjunction with the commonly used vaccine adjuvant alum. Such treatments were also observed to increase nonspecific IL-2, IFN-𝛾, and IL-10 cytokine production, resulting in no improvement of T1D onset prevention. In another approach, we generated a conjugate vaccine by conjugating islet autoantigens to the immunogenic carrier protein, Keyhole Limpet Hemocyanin (KLH). We found that islet antigen-KLH (Ag-KLH) vaccination resulted in significant expansion of the desirable antigen-specific Tregs. Further, Ag-KLH immunization successfully delayed, and in some cases entirely prevented, T1D onset in NOD mice. Indicating that KLH-conjugated vaccine may represent a promising approach for future autoantigen therapies against autoimmunity. Interestingly, administration of anti-IL-7Rα mAbs did not improve these outcomes. To the contrary, we again observed excessive nonspecific cytokine production induced by IL-7Rα blockade that inhibited the beneficial effects of Ag-KLH vaccination. Taken together, we concluded that the addition of anti-IL-7Rα mAbs did not improve the efficacy of autoantigen vaccinations to prevent T1D. Significant work still remains to better characterize and isolate the beneficial effects of anti-IL-7Rα mAbs to treat autoimmunity.