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Natural killer (NK)-like lymphocytes have recently been identified in thymectomised (Tx) Xenopus which are capable of spontaneous cytotoxicity towards the MHC- deficient, allogeneic thymus tumour cell line B(_3)B(_7). This Thesis describes attempts to raise antibodies to Xenopus NK cell surface antigens by phage display and hybridoma technology. The phage display technique was optimised for raising antibodies to novel, cellular antigens in a trial run using the Xenopus thymus tumour cell line B(_3)B(_7). Having isolated a phage antibody which was shown by flow cytometry to bind B(_3)B(_7) cells, the technique was then used to try and raise antibodies to Xenopus NK cells. Isolation of an NIC-specific phage antibody was not achieved but phage antibody XL-6 was raised, which bound an antigen on Xenopus lymphocytes. Phage antibody XL-6, and soluble scFv derived from this, were able to identify a putative mature T cell population in the thymus and may be specific for an amphibian homologue of the mammalian leukocyte common antigen CD45. Hybridoma technology was used to isolate three monoclonal antibodies, 1F8, 4D4 and 1G5, which were shown by flow cytometric analysis to identify a putative NK cell population in control and Tx Xenopus. Following immunomagnetic purification, 1F8- positive spleen cells from control and Tx animals were shown to kill the MHC- deficient tumour target B(_3)B(_7), confirming that this antibody was specific for Xenopus NK cells. Western blotting experiments showed that 1F8, 4D4 and 1G5 identified a doublet of protein bands at 72 and 74 kilodaltons in Xenopus gut lymphoid lysates. Initial attempts to isolate cDNA encoding a Xenopus NK cell surface antigen through immunoscreening a xenopus gut cDNA expression library with antibody 1G5 were unsuccessful as was an attempt to clone a Xenopus homologue of the mammalian NK receptor NKR-Pl by PGR.

No description available.

The large-scale production of monoclonal antibodies (MAb) by mammalian cells in batch and fed-batch culture systems is limited by the unwanted decline in cell viability and reduced productivity that may result from changes in culture conditions. Therefore, it becomes imperative to gain an in-depth knowledge of the factors affecting cell growth and cell viability that in turn determine the antibody production. An attempt has been made to obtain an overall model that predicts the behaviour of both batch and fed-batch systems as a function of the extra-cellular nutrient/metabolite concentrations. Such model formulation will aid in identifying and eventually controlling the dominant factors in play to optimize monoclonal antibody (MAb) production in the future. Murine hybridoma 130-8F producing anti-F-glycoprotein monoclonal antibody was grown in D-MEM medium (Gibco 12100) with 2% FBS. A systematic approach based on Metabolic Flux Analysis (MFA) was applied for the calculation of intracellular fluxes for metabolites from available extracellular concentration values. Based on the set of identified significant fluxes (from MFA), the original metabolic network was reduced to a set of significant reactions. The reactions in the reduced metabolic network were then combined to yield a set of macro-reactions obeying Monod kinetics. Half saturation constants were fixed empirically to avoid computational difficulties that parameter estimation for an over-parameterized system of equations would cause. Using Quadratic Programming, the proposed Dynamic Model was calibrated and model prediction was carried out individually for batch and fed-batch runs. Flux distribution for batch and fedbatch modes were compared to determine whether the same model structure could be applied to both the feeding profiles. Correlation analysis was performed to formulate a Biomass Model for predicting cell concentration and viability as a function of the extracellular metabolite concentrations in batch and fed-batch experiments. Quadratic Programming was applied once again for estimation of growth and death coefficients in the equations for viable and dead cell predictions. The prediction accuracy of these model equations was tested by using experimental data from additional runs. Further, the Dynamic Model was integrated with the Biomass Model to get an Integrated Model capable of predicting concentration values for substrates, extracellular metabolites, and viable and dead cell concentration by utilizing only starting concentrations as input. It was found that even though the set of significant fluxes was the same for batch and fedbatch operations, the order of these fluxes was different between the two systems. There was a gradual metabolic shift in the fed-batch system with time indicating that under conditions of nutrient limitation, the available energy is channeled towards maintenance rather than growth. Also, available literature with regard to cell kinetics during fed-batch operation suggests that under nutrient limited conditions, the cells move from a viable, non-apoptotic state to a viable apoptotic state. This is believed to lead to variations in antibody production rates and might explain inaccurate predictions for MAb obtained from the model proposed in the current work. As a result more detailed analysis of the system and in particular, the switch from non-apoptotic to apoptotic state is required. As a continuation of efforts to study the system in-depth, fluorescence imaging is currently being applied as a tool to capture the changes in cell morphology along the course of experimental batch and fed-batch runs. These experiments maybe able to elucidate the transition from non-apoptotic to apoptotic cells and this information maybe used in the future to improve the accuracy of the existing mathematical model.

The large-scale production of monoclonal antibodies (MAb) by mammalian cells in batch and fed-batch culture systems is limited by the unwanted decline in cell viability and reduced productivity that may result from changes in culture conditions. Therefore, it becomes imperative to gain an in-depth knowledge of the factors affecting cell growth and cell viability that in turn determine the antibody production. An attempt has been made to obtain an overall model that predicts the behaviour of both batch and fed-batch systems as a function of the extra-cellular nutrient/metabolite concentrations. Such model formulation will aid in identifying and eventually controlling the dominant factors in play to optimize monoclonal antibody (MAb) production in the future. Murine hybridoma 130-8F producing anti-F-glycoprotein monoclonal antibody was grown in D-MEM medium (Gibco 12100) with 2% FBS. A systematic approach based on Metabolic Flux Analysis (MFA) was applied for the calculation of intracellular fluxes for metabolites from available extracellular concentration values. Based on the set of identified significant fluxes (from MFA), the original metabolic network was reduced to a set of significant reactions. The reactions in the reduced metabolic network were then combined to yield a set of macro-reactions obeying Monod kinetics. Half saturation constants were fixed empirically to avoid computational difficulties that parameter estimation for an over-parameterized system of equations would cause. Using Quadratic Programming, the proposed Dynamic Model was calibrated and model prediction was carried out individually for batch and fed-batch runs. Flux distribution for batch and fedbatch modes were compared to determine whether the same model structure could be applied to both the feeding profiles. Correlation analysis was performed to formulate a Biomass Model for predicting cell concentration and viability as a function of the extracellular metabolite concentrations in batch and fed-batch experiments. Quadratic Programming was applied once again for estimation of growth and death coefficients in the equations for viable and dead cell predictions. The prediction accuracy of these model equations was tested by using experimental data from additional runs. Further, the Dynamic Model was integrated with the Biomass Model to get an Integrated Model capable of predicting concentration values for substrates, extracellular metabolites, and viable and dead cell concentration by utilizing only starting concentrations as input. It was found that even though the set of significant fluxes was the same for batch and fedbatch operations, the order of these fluxes was different between the two systems. There was a gradual metabolic shift in the fed-batch system with time indicating that under conditions of nutrient limitation, the available energy is channeled towards maintenance rather than growth. Also, available literature with regard to cell kinetics during fed-batch operation suggests that under nutrient limited conditions, the cells move from a viable, non-apoptotic state to a viable apoptotic state. This is believed to lead to variations in antibody production rates and might explain inaccurate predictions for MAb obtained from the model proposed in the current work. As a result more detailed analysis of the system and in particular, the switch from non-apoptotic to apoptotic state is required. As a continuation of efforts to study the system in-depth, fluorescence imaging is currently being applied as a tool to capture the changes in cell morphology along the course of experimental batch and fed-batch runs. These experiments maybe able to elucidate the transition from non-apoptotic to apoptotic cells and this information maybe used in the future to improve the accuracy of the existing mathematical model.

The groupers are vital fish in the market of over 350 million dollars, while grouper nervous necrosis virus (NNV) has caused mass mortality at about 100% in larvae and juveniles, which impacts on economic of marine cultured fish. The monoclonal antibody is one of the best methods to identify the epitopes on the 3D structure. For evaluation, the Balb/c mice were injected with DGNNV and virus-like particles (VLPs) in this study. The results showed that ascite of mAb-cells produced 1200 times higher than the cell secretion in the medium whereas our best clone hAb_VLP8 can only produced 100 times less antibody than the cell secretion. In the meantime before the monoclonal producer is established, the hAb_VLP8 could be used for ascite production to gain high antibody production.

The aim of this master‘s thesis is to prepare a monoclonal antibody against cyclin-dependent kinase 13 (CDK13). The theoretical part focuses on antigen-antibody binding, which is essential for the use of monoclonal antibodies in the determination of CDK13 as well as the transcription that this kinase affects. This section is also devoted to Western blot and ELISA methods for detection of newly generated antibodies. Furthermore, the antibodies and the antigen definition are stated, which are later on discussed. The practical part is devoted to the preparation of antigen - its isolation and purification on a peristaltic pump. It also addresses immunization, its course, and the amount of antigen used to immunize mice. After immunization, the work focuses on fusion of sp-2 cells and splenocytes, which were first removed from the immunized mouse and purified. After the fusion alone, selection of hybridomas on HAT selection medium is mentioned, followed by detection first by ELISA and later by Western blotting. The resulting hybridomas with positive ELISA response are frozen for further testing at the Veterinary Research Institute in Brno, where the entire practical part of this thesis was carried out. These frozen hybridomas are further tested by immunoprecipitation to conclude this thesis.

Monoclonal antibodies are powerful and versatile tools that enable the study of proteins in diverse contexts. They are often utilized to assist with identification of subcellular localization and characterization of the function of target proteins of interest. However, because there can be considerable sequence diversity between orthologous proteins in Xenopus and mammals, antibodies produced against mouse or human proteins often do not recognize Xenopus counterparts. To address this issue, we refined existing mouse monoclonal antibody production protocols to generate antibodies against Xenopus proteins of interest. Here, we describe several approaches for the generation of useful mouse anti-Xenopus antibodies to multiple Xenopus proteins and their validation in various experimental approaches. These novel antibodies are now available to the research community through the Developmental Study Hybridoma Bank (DSHB).

No description available.

Actuellement, 60% des médicaments sur le marché ont pour cible des protéines membranaires. Toutefois, l'étude de ces protéines membranaires reste un challenge de par leur structure particulière (domaines transmembranaires hydrophobes et domaines extra- et intra-cellulaires hydrophiles), mais également par leur faible expression sur les cellules.L'entreprise Ciloa, dans laquelle j'ai effectué ma thèse, a développé une technologie brevetée, qui permet d'exprimer à la surface des exosomes, des vésicules membranaires de tailles comprises entre 30 et 100nm, des protéines membranaires natives, grâce à un peptide d'adressage, le DCTM. Cette technologie possède de nombreux domaines d'applications, comme le criblage de médicaments, le développement de vaccins ou encore le développement d'anticorps monoclonaux.L'objectif de ma thèse a été, dans un premier temps, de mettre en place l'outil exosomes recombinants grâce à la technologie de Ciloa et dans un deuxième temps, d'utiliser ces outils pour le développement d'anticorps, grâce aux exosomes recombinants.Ainsi, j'ai d'abord mis au point différentes techniques de caractérisation des exosomes recombinants (ELISA), et également participé à la mise en place de différents protocoles de production et de purification, en fonction leur utilisation. Une fois ces outils optimisés, j'ai pu les utiliser pour le développement d'anticorps. J'ai testé en parallèle deux méthodes de production d'anticorps, une méthode classique, l'hybridation lymphocytaire après immunisation de souris BALB/c, et une méthode plus récente, le criblage d'une banque de scFvs par phage display.L'hybridation lymphocytaire a permis la production d'hybridomes, dont les anticorps ont été criblés sur exosomes, par ELISA. Dans le cadre du criblage par phage display, j'ai participé au développement d'une banque de scFvs, basée sur le modèle du 13R4, dont nous avons modifié les longueurs de boucles des différents CDRs, notamment le CDRH3, afin de cibler les épitopes faiblement accessibles des protéines membranaires. Les sélections de scFvs ont été effectuées sur exosomes recombinants, exprimant des protéines membranaires. / Nowadays, more than 60% of marketed drugs target membrane proteins. However, their study still represents a challenge, essentially due to their particular 3D-structure (hydrophobic transmembrane domains and hydrophilic extra- and intra-cellular domains), but also to their low expression level in cells.Ciloa, the start-up company in which I realized my PhD, has developed a patented technology that enables to express native membrane proteins on exosomes, membrane vesicles of 30 to 100nm, using a pilot peptide called DCTM (for Cytosolic Domain of TransMembrane). This technology displays a lot of different applications, in different domains such as drug screening, vaccines development or monoclonal antibodies (mAbs) development.The purpose of my PhD research was, first, to set up the recombinant exosomal tool using Ciloa's innovative technology, and then to use this tool to develop monoclonal antibodies.Thus, at the beginning of my PhD, I set up exosomal characterization technics, such as ELISA, and I also took part in the setup of several production and purification protocols, depending of the use of exosomes. Once these tools had been optimized, I was able to use them to develop mAbs. I tested two methods, one classical, the generation of hybridoma after Balb/c mice immunizations, and a more recent technology, the screening of scFvs library by phage display.Therefore, I obtained hybridoma and was able to screen the derived antibodies by ELISA on exosomes. Concerning the phage display technology, I took part in the development of a new scFvs library, based on the 13R4 scaffold, of which we changed the CDRs lengths, mostly the CDRH3, in order to target epitopes with low accessibility, such as the one of membrane proteins. The library screening was realized on recombinant exosomes.