Die vorliegende Arbeit wurde im Zeitraum von Oktober 2002 bis November 2005 an dem Institut für Biochemie und Biologie der Universität Potsdam in Kooperation mit dem Institut für Chemie des GKSS Forschungszentrums in Teltow unter der Leitung von Herrn Prof. Dr. B. Micheel und Herrn Prof. Dr. Th. Groth angefertigt. Im Rahmen dieser Arbeit wurden die Wechselwirkungen von Immunzellen mit verschiedenen Kultursubstraten untersucht. Dafür wurden drei verschiedene Hybridomzelllinien eingesetzt. Eine Hybridomzelllinie (K2) ist im Laufe dieser Arbeit hergestellt und etabliert worden. <br><br> Der Einsatz von synthetischen und proteinbeschichteten Kulturoberflächen führte bei Hybridomzellen zu einer deutlich gesteigerten Antikörpersynthese im Vergleich zu herkömmlichen Zellkulturmaterialien. Obwohl diese Zellen in der Regel als Suspensionszellen kultiviert werden, führten die eingesetzten Polymermembranen (PAN, NVP) zu einer verbesserten Antikörpersynthese (um 30%) gegenüber Polystyrol als Referenz. Es konnte gezeigt werden, dass es einen Zusammenhang zwischen der Produktivität und dem Adh asionsverhalten der Hybridomzellen gibt. <br><br> Um den Einfluss von Proteinen der extrazellulären Matrix auf Zellwachstum und Antikörpersynthese von Hybridomzellen zu untersuchen, wurden proteinbeschichtete Polystyrol-Oberflächen eingesetzt. Für die Modifikationen wurden Fibronektin, Kollagen I, Laminin und BSA ausgewählt. Die Modifikation der Polystyrol-Oberfläche mit geringen Mengen Fibronektin (0,2-0,4 µg/ml) führte zu einer beträchtlichen Steigerung der Antikörpersynthese um 70-120%. Für Kollagen I- und BSA-Beschichtungen konnten Steigerungen von 40% beobachtet werden. Modifikationen der Polystyrol-Oberfläche mit Laminin zeigten nur marginale Effekte. Durch weitere Versuche wurde bestätigt, dass die Adhäsion der Zellen an Kollagen I- und Laminin-beschichteten Oberflächen verringert ist. Die alpha2-Kette des alpha2beta1-Integrins konnte auf der Zelloberfläche nicht nachgewiesen werden. Durch ihr Fehlen wird wahrscheinlich die Bindungsfähigkeit der Zellen an Kollagen I und Laminin beeinflusst. Durch die Ergebnisse konnte gezeigt werden, dass Hybridomzellen nicht nur Suspensionszellen sind und das Kultursubstrate das Zellwachstum und die Produktivität dieser Zellen stark beeinflussen können. Der Einsatz von synthetischen und proteinbeschichteten Kultursubstraten zur Steigerung der Antikörpersynthese kann damit für die industrielle Anwendung von großer Relevanz sein. Für die Modellierung einer Lymphknotenmatrix wurden Fibronektin, Kollagen I, Heparansulfat und N-Acetylglucosamin-mannose in verschiedenen Kombinationen an Glasoberflächen adsorbiert und für Versuche zur In-vitro-Immunisierung eingesetzt. Es konnte gezeigt werden, dass die Modifikation der Oberflächen die Aktivierung und Interaktion von dendritischen Zellen, T- und B-Lymphozyten begünstigt, was durch den Nachweis spezifischer Interleukine (IL12, IL6) und durch die Synthese spezifischer Antikörper bestätigt wurde. Eine spezifische Immunreaktion gegen das Antigen Ovalbumin konnte mit den eingesetzten Zellpopulationen aus Ovalbumin-T-Zell-Rezeptor-transgenen Mäusen nachgewiesen werden. Die In-vitro-Immunantwort wurde dabei am stärksten durch eine Kombination von Kollagen I, Heparansulfat und N-Acetylglucosamin-mannose auf einer Glasoberfläche gefördert. Die Etablierung einer künstlichen Immunreaktion kann eine gesteuerte Aktivierung bzw. Inaktivierung von körpereigenen dendritischen Zellen gegen bestehende Krankheitsmerkmale in vitro ermöglichen. Durch die Versuche wurden Grundlagen für spezifische Immunantworten erarbeitet, die u.a. für die Herstellung von humanen Antikörpern eingesetzt werden können. / In this scientific work the interactions of immune cells with different culture substrata were investigated. Therefore, three hybridoma cell lines were tested, one cell line (K2) was established during this work. The application of synthetic and protein-coated culture surfaces lead to a significantly increased synthesis of monoclonal antibodies in comparison to usual tissue polystyrene. Although hybridoma cells were normally cultured in suspension applied polymer membranes like PAN and NVP induced an increase by 30%. Furthermore, an influence of cell adhesion and antibody synthesis could be shown. To investigate the influence of extracellular matrix proteins on growth and antibody synthesis of hybridoma cells tissue culture polystyrene was coated with fibronectin, collagen I, laminin and bovine serum albumine in different concentrations. Modifications with fibronectin (concentrations between 0.2 and 0.4 µg/ml) improved the yield of monoclonal antibodies considerably by 70-120%. Coating cell culture plates with collagen I and bovine serum albumine induced an increase by 40%. The coating with laminin showed only marginal effects. Further experiments approved a decreased adhesion of hybridoma cells on collagen I and laminin coated surfaces. FACS analysis showed a reduced presence of the alpha2-chain of the alpha2/beta1-integrin responsible for mediating the binding to collagen I and laminin. Probably, the binding affinity to collagen I and laminin coated surfaces was influenced by this. The results showed a high impact of modified culture substrata on antibody synthesis even if hybridoma cells were cultured in suspension normally and this could be an approach for industrial application. The second part of this work comprised the creation of a lymph node paracortex related surface. Different matrix proteins like fibronectin, collagen I, heparane sulfate and a sugar named N-acetylglucosamine-mannose were coated in different combinations on glass surfaces to create a matrix. Dendritic cells were cultivated on these surfaces and get activated with ovalbumin. After that naïve T- and B-cell populations were added and it could be shown nicely that the modifications of the culture surface were essential for activation and interaction of dendritic cells, T- and B-cells which resulted in the secretion of specific interleukins (IL12, IL6) and specific antibodies (anti-ovalbumin-antibodies). In these experiments a specific immune respone to ovalbumin in vitro could be detected if the cells were isolated from ovalbumin-receptor-transgenic-mice (TgNDO11.10). This In-vitro-immunization was triggered at most if cells were cultured on a surface coated with a combination of collagen I, heparane sulfate and N-acetylglucosamine-mannose. These experiments could be basics for controlled specific immune reactions in vitro which could be used for the production of human antibodies or for the controlled activation or inactivation of immune cells.

Grouper in Taiwan is of high value, but nervous necrosis virus infection causes 100% mortality. Our laboratory had developed a good expression system to produce virus-like particles that induced immune functions. In this study, cells producing monoclonal antibody against the virus-like particles were used to induce BALB/c mice production of high titer ascites. The ascite from the H1 cells generated 6000-fold of antibodies higher than cell culture, using enzyme-linked immune-sorbent assay, although relatively less than the previous monoclonal cells of mX, 8000-fold. In the electron microscopy, the ascite antibody bound to various mutants of virus-like particles. Using the polymerase chain reaction to amplify fragments of IgG cDNAs, we will clone and express such cDNAs to efficiently produce the desired monoclonal antibodies.

DNA damage occurs through endogenous and exogenous sources, and can lead to stalled replication forks, genetic disorders, cancer, and cell death. Homologous recombination (HR) is a relatively fast and error-free repair pathway for damaged DNA, which can occur through a gene conversion event or through a crossing-over event with the exchange of genetic material. Homologous recombination occurs most frequently in the G2 phase of the cell cycle and utilizes the sister chromatid as the repair template. When the sister chromatid is unavailable, the homologous chromosome or a homologous sequence in an ectopic location can be used to repair the lesion; the latter of which is referred to as ectopic homologous recombination (EHR). Rad51 is a key protein involved in HR, and to test its role in EHR, variant Rad51 proteins were expressed in murine hybridoma cells. These Rad51 variants were assayed for their effects on EHR. Excess wild-type Rad51 as well as a deficiency of wild-type Rad51 decreased EHR from the background level found in these cell lines. Thus, Rad51 is necessary for EHR, but there may be an optimal amount of Rad51 required for efficient EHR. Expression of the Rad51 catalytic mutants Rad51K133A and Rad51K133R was found to have an inhibitory effect on EHR, as expected based on the loss of ATP binding and ATP hydrolysis, respectively, in these variants. Excess wild-type Rad51 was verified in this study to increase HR via a gene targeting assay. MMC treatment, but not ionizing radiation, leads to an increase in EHR in the presence of excess wild-type Rad51. Thus, endogenous levels of Rad51 are sufficient to maintain EHR, but in the presence of excess wild-type Rad51, the level of EHR can increase in response to certain DNA damaging agents and in response to gene targeting.

No description available.

Aim of this diploma thesis was to prepare two protein antigens and two monoclonal antibodies, all based on VP2 minor protein of human polyomaviruses BK virus and Merkel Cell Polyomavirus. One monoclonal antibody was being prepared against unique part of VP2 protein (N-terminal epitope, not present in VP3 protein). A cell line producing such monoclonal antibody has never been established before due to low immunogenicity of the epitope. Our approach was successful in terms of mouse immunization, however, serious problems with hybridoma line stability appeared later during the preparation process. Preparation of antibody targeted to the sequence of VP2 protein of Merkel Cell Polyomavirus was another aim of this thesis. Mouse immunization and hybridoma fusion were performed successfully. After four rounds of cloning in order to purify an established clone, nine clones were cultivated in larger scale. This cultivation probably led to diminished antibody specificity and loss of production ability in most of the hybridoma cells. One more cloning should give rise to an established clone with sufficient production. Two preparations of protein antigens were performed in two expression systems. DNA encoding C-terminally truncated protein VP2 of BK virus fused with His-tag was cloned into a vector suitable for...

A contaminação da água para consumo humano por toxinas produzidas por cianobactérias é um problema de saúde pública e das autoridades em todo o mundo. Microcistina-LR (MCLR) é uma cianotoxina heptapeptídica cíclica que inibe as proteínas fosfatases PP1 E PP2A nos hepatócitos. Microcistinas são produzidas por diversos gêneros de cianobactérias e mais de 70 variações estruturais têm sido caracterizadas em florações naturais. Por serem haptenos, as microcistinas são incapazes de induzir uma resposta imune em animais. Conseqüentemente, foi necessário aplicar métodos de conjugação envolvendo a adição de uma proteína carreadora, mcKLH (cationized Keyhole Limpet Hemocyanin). Portanto, o objetivo inicial desta tese foi o de obter anticorpos monoclonal (em camundongos) e policlonal (em coelho) anti- MCLR. Com relação ao anticorpo monoclonal foram obtidos 9 hibridomas (k29, k210, k317, k248, k284, k290, k2161, k2226, k2232), sendo que apenas 5 se mostraram estáveis (k29, k317, k248, k284, k2232). Estes foram selecionados para serem isotipados, expandidos em líquido ascítico, purificados em coluna cromatográfica de proteína-A e titulados. Dentre estes cinco hibridomas secretores de anticorpos, o clone k317 foi o que melhor reconheceu (mais específico) a toxina MCLR. Os anticorpos do sobrenadante de meio de cultura do hibridoma e o fluido ascítico purificado foram identificados pelo ensaio ELISA (Enzyme Linked Immunosorbent Assay) previamente padronizado. Mesmo sensibilizando a placa de ELISA com diferentes antígenos, tais como MCLR-cBSA, MCLR, MCLR, MCRR, MCYR e MCLA, o clone 17 foi o que apresentou melhor linearidade frente às variantes de microcistina. Portanto, o clone 17 (isótipo IgG1) obtido é muito promissor e será usado para detecção de MCLR na água para consumo humano através do desenvolvimento de um kit de ELISA competição. Com relação ao anticorpo policlonal, o antígeno de imunização foi MCLR-mcKLH, enquanto que o antígeno de sensibilização foi MCLR-cBSA para o ensaio de titulação de anticorpos de classe IgG por ELISA indireto. Na seqüencia, foi padronizado um ensaio ELISA competição utilizando somente a toxina MCLR como antígeno de sensibilização. Este método Caseína foi padronizado, validado e comparado com o kit comercial Abraxis®. O kit ELISA competição que utiliza anticorpo policlonal, nomeado como método Caseína, foi avaliado quanto Limite Inferior de Quantificação, Especificidade, Seletividade, influência do metanol no ensaio, Recuperação, Linearidade, Precisão, Exatidão e Robustez. Este método de triagem apresentou excelente resultado quando comparado ao kit comercial Abraxis®, pois foi capaz de detectar tanto variantes de microcistinas como nodularinas no ambiente aquático. O ensaio ELISA competição utilizando anticorpo policlonal anti-MCLR foi submetido à patente pela Agência USP de Inovação (I.N.P.I. 018090046230). / The contamination of drinking water by cyanobacterial toxins is a public health issue and a concern for water authorities throughout the world. Microcystin-LR (MCLR) is a hazardous cyclic heptapeptide cyanotoxin, which inhibits protein phosphatase PP1 and PP2A in hepatocytes. Microcystins are produced by several genera of cyanobacteria and presents more than 70 structural variations characterized in natural blooms. As haptens, microcystins are unable to invoke an immune response in animals. Consequently, the application of conjugation methods with an additional carrier protein, the KLH (Keyhole Limpet Hemocyanin) was necessary. The main objective of this study was to obtain monoclonal (in mice) and polyclonal (in rabbits) antibodies for reacting against MCLR. In what refers to monoclonal antibodies, 9 hybridomas (k29, k210, k317, k248, k284, k290, k2161, k2226, k2232) were obtained; however only 5 were stables (k29, k317, k248, k284, k2232). These were selected to be isotyped, expanded in ascitic fluid, purified by protein-A column chromatography and then, they were titrated. Out of these five antibody-secretor hybridomas, clone k317 was the best to recognize (more specific) the MCLR toxins. Antibodies in hybridoma cell culture supernatant and purified ascites fluid were identified by ELISA assay (Enzyme Linked Immunosorbent Assay) as prior standardized. Even when sensitizing ELISA plate with different antigens, as MCLR-cBSA, MCLR, MCLR, MCRR, MCYR and MCLA, clone 17 presented the best linearity against microcystin variants. Therefore, the obtained clone 17 (isotype IgG1) is a promising clone and shall be used for detecting MCLR in drinking water through the development of a competitive ELISA immunoassay kit. In what refers to the polyclonal antibody, MCLR-mcKLH was used as immunization antigen, while MCLR-cBSA was used as sensitizing antigen for the IgG titration assay by indirect ELISA. In the sequence, a competition ELISA assay was standardized using the MCLR toxin as sensitizing antigen. This Casein method was standardized, validated and compared to the commercial kit Abraxis®. The competition ELISA kit using polyclonal antibody, known as Casein method, was analyzed concerning its Quantification Inferior Limit, Specificity, Selectivity, methanol influence of the assay, Recuperation, Linearity, Precision, Accuracy and Robustness. This screening method reached excellent results if compared to the commercial kit Abraxis®, for being able to detect both the microcystins variants and the nodularins in aquatic environmental. The competition ELISA assay using anti-MCLR polyclonal antibody was submitted to the grant of a patent by USP Innovation Agency (INPI 018090046230).

Increasing demand on more effective cell culture reactors has driven optimization works to increase output of products. This has led to development of soft sensors that uses mathematical formulas to increase the available information for the parameters during runs. In the project two parameters was evaluated for use in such a soft sensor, viability by measuring on-line capacitance with Aber probe and L-lactate production using BioSenz apparatus. To determine how well these could be used both were used on batch reactors measuring on a mouse-mouse B cell hybridoma culture which produced IgG1. On-line measurements were performed by probes which measured directly on the cell suspension or withdrew sterile sample from the reactor. Measuring viability gave results with low error, which can be concluded to the variation in reference cell count, but it could not be determined if measuring L-lactate production with BioSenz works in reactors of this size. More work needs to be done on other types of reactors, like fed-batch or perfusion, or lower working volumes.

A contaminação da água para consumo humano por toxinas produzidas por cianobactérias é um problema de saúde pública e das autoridades em todo o mundo. Microcistina-LR (MCLR) é uma cianotoxina heptapeptídica cíclica que inibe as proteínas fosfatases PP1 E PP2A nos hepatócitos. Microcistinas são produzidas por diversos gêneros de cianobactérias e mais de 70 variações estruturais têm sido caracterizadas em florações naturais. Por serem haptenos, as microcistinas são incapazes de induzir uma resposta imune em animais. Conseqüentemente, foi necessário aplicar métodos de conjugação envolvendo a adição de uma proteína carreadora, mcKLH (cationized Keyhole Limpet Hemocyanin). Portanto, o objetivo inicial desta tese foi o de obter anticorpos monoclonal (em camundongos) e policlonal (em coelho) anti- MCLR. Com relação ao anticorpo monoclonal foram obtidos 9 hibridomas (k29, k210, k317, k248, k284, k290, k2161, k2226, k2232), sendo que apenas 5 se mostraram estáveis (k29, k317, k248, k284, k2232). Estes foram selecionados para serem isotipados, expandidos em líquido ascítico, purificados em coluna cromatográfica de proteína-A e titulados. Dentre estes cinco hibridomas secretores de anticorpos, o clone k317 foi o que melhor reconheceu (mais específico) a toxina MCLR. Os anticorpos do sobrenadante de meio de cultura do hibridoma e o fluido ascítico purificado foram identificados pelo ensaio ELISA (Enzyme Linked Immunosorbent Assay) previamente padronizado. Mesmo sensibilizando a placa de ELISA com diferentes antígenos, tais como MCLR-cBSA, MCLR, MCLR, MCRR, MCYR e MCLA, o clone 17 foi o que apresentou melhor linearidade frente às variantes de microcistina. Portanto, o clone 17 (isótipo IgG1) obtido é muito promissor e será usado para detecção de MCLR na água para consumo humano através do desenvolvimento de um kit de ELISA competição. Com relação ao anticorpo policlonal, o antígeno de imunização foi MCLR-mcKLH, enquanto que o antígeno de sensibilização foi MCLR-cBSA para o ensaio de titulação de anticorpos de classe IgG por ELISA indireto. Na seqüencia, foi padronizado um ensaio ELISA competição utilizando somente a toxina MCLR como antígeno de sensibilização. Este método Caseína foi padronizado, validado e comparado com o kit comercial Abraxis®. O kit ELISA competição que utiliza anticorpo policlonal, nomeado como método Caseína, foi avaliado quanto Limite Inferior de Quantificação, Especificidade, Seletividade, influência do metanol no ensaio, Recuperação, Linearidade, Precisão, Exatidão e Robustez. Este método de triagem apresentou excelente resultado quando comparado ao kit comercial Abraxis®, pois foi capaz de detectar tanto variantes de microcistinas como nodularinas no ambiente aquático. O ensaio ELISA competição utilizando anticorpo policlonal anti-MCLR foi submetido à patente pela Agência USP de Inovação (I.N.P.I. 018090046230). / The contamination of drinking water by cyanobacterial toxins is a public health issue and a concern for water authorities throughout the world. Microcystin-LR (MCLR) is a hazardous cyclic heptapeptide cyanotoxin, which inhibits protein phosphatase PP1 and PP2A in hepatocytes. Microcystins are produced by several genera of cyanobacteria and presents more than 70 structural variations characterized in natural blooms. As haptens, microcystins are unable to invoke an immune response in animals. Consequently, the application of conjugation methods with an additional carrier protein, the KLH (Keyhole Limpet Hemocyanin) was necessary. The main objective of this study was to obtain monoclonal (in mice) and polyclonal (in rabbits) antibodies for reacting against MCLR. In what refers to monoclonal antibodies, 9 hybridomas (k29, k210, k317, k248, k284, k290, k2161, k2226, k2232) were obtained; however only 5 were stables (k29, k317, k248, k284, k2232). These were selected to be isotyped, expanded in ascitic fluid, purified by protein-A column chromatography and then, they were titrated. Out of these five antibody-secretor hybridomas, clone k317 was the best to recognize (more specific) the MCLR toxins. Antibodies in hybridoma cell culture supernatant and purified ascites fluid were identified by ELISA assay (Enzyme Linked Immunosorbent Assay) as prior standardized. Even when sensitizing ELISA plate with different antigens, as MCLR-cBSA, MCLR, MCLR, MCRR, MCYR and MCLA, clone 17 presented the best linearity against microcystin variants. Therefore, the obtained clone 17 (isotype IgG1) is a promising clone and shall be used for detecting MCLR in drinking water through the development of a competitive ELISA immunoassay kit. In what refers to the polyclonal antibody, MCLR-mcKLH was used as immunization antigen, while MCLR-cBSA was used as sensitizing antigen for the IgG titration assay by indirect ELISA. In the sequence, a competition ELISA assay was standardized using the MCLR toxin as sensitizing antigen. This Casein method was standardized, validated and compared to the commercial kit Abraxis®. The competition ELISA kit using polyclonal antibody, known as Casein method, was analyzed concerning its Quantification Inferior Limit, Specificity, Selectivity, methanol influence of the assay, Recuperation, Linearity, Precision, Accuracy and Robustness. This screening method reached excellent results if compared to the commercial kit Abraxis®, for being able to detect both the microcystins variants and the nodularins in aquatic environmental. The competition ELISA assay using anti-MCLR polyclonal antibody was submitted to the grant of a patent by USP Innovation Agency (INPI 018090046230).

Un problème fondamental pendant l'évaluation in vitro de la toxicité de composés organiques volatils (COVs) est le manque de connaissance de l'évolution de la concentration des COVs à laquelle les systèmes vivants sont exposés au cours des études expérimentales. Ce travail présente un nouveau dispositif expérimental conçu pour étudier l'exposition des systèmes vivants aux COVs. Le dispositif est formé de deux compartiments séparés par une membrane hydrophobe poreuse et permet des durées relativement longues de manipulations sans restreindre la respiration cellulaire. Une modélisation théorique qui couple la conservation de masse et du moment entre les différentes phases et la respiration des cellules hybridomes (ATCC CRL-1606) au sein du dispositif a été développée. Le modèle permet de prédire l'évolution de la concentration des COVs, de l'oxygène et du dioxyde de carbone dans le dispositif. Les résultats simulés pour le transfert des COVs ont revélé une bonne concordance avec les résultats expérimentaux et ont montré que le type de membrane et son diamètre, le coefficient de partage des COVs et la hauteur de la phase liquide ont une influence significative sur l'évolution de la concentration de ceux-ci dans la phase liquide. Néanmoins la disponibilité de l'oxygène au niveau des cellules dépend principalement de la densité cellulaire initiale, de la vitesse spécifique de consommation de ce gaz et de la hauteur du liquide alors que les paramètres liés à la membrane ont une influence sur le contrôle du pH. / A major problem during in vitro evaluation of the toxicity of volatile organic compounds (VOC) is the lack of knowledge of the evolution of the concentration of such compounds during the course of experimental studies with living systems. This work presents the design of a novel experimental device for the study of cell culture exposure to VOCs. The device is made of two compartments separated by a porous hydrophobic membrane and allows relatively long durations of handling without restricting cellular breathing. A theoretical modeling which couples mass and moment conservation between the different phases inside the device with the breathing kinetics of hybridoma cells (ATCC CRL-1606) was developed. The model allows predicting the evolution of the concentration of the VOCs, the oxygen and the carbon dioxide inside the device. The simulations of the mass transfer of the VOCs simulated presented a good agreement with experiments and showed that the type of membrane and its diameter, the VOCs partition coefficient and the height of the liquid phase have a significant influence on the evolution of their concentration in the liquid phase. Nevertheless, the availability of oxygen for the cells depends mainly on the initial cellular density, the specific kinetics of consumption of this gas and on the height of the liquid phase, whereas the parameters related to membrane have an influence on the control of the pH.

Submitted by Aelson Maciera (aelsoncm@terra.com.br) on 2017-10-06T18:08:45Z No. of bitstreams: 1 DissACP.pdf: 6605106 bytes, checksum: c63dcdec106579e271edaa4dc5a8bdcf (MD5) / Approved for entry into archive by Ronildo Prado (bco.producao.intelectual@gmail.com) on 2017-11-28T12:14:39Z (GMT) No. of bitstreams: 1 DissACP.pdf: 6605106 bytes, checksum: c63dcdec106579e271edaa4dc5a8bdcf (MD5) / Approved for entry into archive by Ronildo Prado (bco.producao.intelectual@gmail.com) on 2017-11-28T12:14:55Z (GMT) No. of bitstreams: 1 DissACP.pdf: 6605106 bytes, checksum: c63dcdec106579e271edaa4dc5a8bdcf (MD5) / Made available in DSpace on 2017-11-28T12:26:25Z (GMT). No. of bitstreams: 1 DissACP.pdf: 6605106 bytes, checksum: c63dcdec106579e271edaa4dc5a8bdcf (MD5) Previous issue date: 2017-02-24 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Nowadays, cancer is one of the most feared diseases, affecting each day more and more people worldwide. The importance of new cancer treatment researches is very clear since the ones that has been used are not very effective and may lead to drug resistance, implying in a constant dose increasing which can lead to toxicity issues. Collateral effects and the instability generated in the patient’s organism are also reasons why the necessity of discovering new cancer treatments is imminent. A treatment alternative that has aroused interest is the use of monoclonal antibodies as immunotherapics, since they act by stimulating the patient’s immune system neutralizing the tumor cells in a very efficient and specific way. This kind of antibody can be produced by culturing hybrid animal cells, better known as hybridoma, under strictly controlled conditions so they can be studied and used in human beings. For this reason, the major goal of this project was the production of murine monoclonal antibodies using hybridoma cell culture in order to stablish an efficient culture methodology for hybridomas PC-61 or DTA1 producers of monoclonal antibodies anti-CD25 and anti-GITR, respectively, with high quality and enough amounts using Fetal Bovine Serum (FBS) free medium to, in the future, carry out animal model studies of their potential as therapeutic agents for cancer treatment. Both hybridomas were cultivated on a small scale with RPMI medium and addition of SFB, for comparative purposes and only one was selcted for the second step. The sequential adaptation methodology test, consisted in a gradual percent’s reduction of medium with serum at the same time that increase the percentage of commercial medium without serum, and was selected the medium without SFB in which the hybridoma was better adapted. After was carried out on a laboratory scale in a system type spinner flask (500 ml) with the commercial medium selected in the previous step, in controlled conditions of temperature (37 ° C) and pH (7.2). Based on analyzes of cell culture results, amino acid consumption and monoclonal antibodies quantification , SFM commercial medium SFB-free provided better results for culturing the PC-61 hybridoma, allowing the pilot scale culture reached even higher cell densities than in the standard medium with addition of FBS. / O câncer é uma das doenças mais temidas da atualidade, e atinge cada vez mais pessoas em todo o mundo. A importância de pesquisas sobre novos tratamentos na luta contra o câncer é clara e consensual, uma vez que os que vem sendo utilizados, não são muito eficientes, causam resistência à medicação utilizada o que implica na utilização de doses crescentes que por sua vez podem gerar problemas de toxicidade. Os efeitos colaterais e a instabilidade gerada no organismo do paciente, também são fatores da necessidade de pesquisar novos caminhos para o tratamento do câncer. Uma das alternativas de tratamento que tem despertado interesse é a utilização de anticorpos monoclonais (mAbs) como imunoterápicos, os quais agem estimulando o próprio sistema imune do paciente neutralizando a ação das células tumorais de forma eficiente e específica. A produção de tais anticorpos pode ser feita mediante o cultivo de células animais híbridas, mais conhecidas como hibridomas, sobre condições estritamente controladas para que possam ser estudados e utilizados em humanos. Por essa razão definiu-se como objetivo desse trabalho a produção anticorpos monoclonais murinos por meio de cultivo de hibridomas com a finalidade de estabelecer uma metodologia eficiente de cultivo dos hibridomas PC-61 ou DTA1 secretores dos mAbs anti-CD25 e antiGITR respectivamente, com qualidade e em quantidades suficientes utilizando meios livres de soro fetal bovino (SFB), para a seguir efetuar estudos em modelo animal de seu potencial como agentes terapêuticos no tratamento de câncer. Os dois hibridomas foram cultivados em pequena escala utilizando meio RPMI-1640 e adição de SFB, para fins comparativos e somente um foi selecionado para a próxima etapa. O teste da metodologia de adaptação sequencial, onde houve a redução gradativa da porcentagem de meio RPMI-1640 com 10% de SFB e o aumento da porcentagem de meio comercial sem SFB, e foi selecionado o meio livre de SFB em que o hibridoma melhor se adaptou. Posteriormente foi realizado o cultivo do hibridoma em escala laboratorial em sistema de frasco agitado biorreator do tipo Spinner (500 mL) com o meio livre de SFB selecionado na etapa anterior, sob condições bem controladas de temperatura (37ºC), pH (7,2). Com base nas análises dos resultados dos cultivos celulares, metabolismo de aminoácidos e quantificação de mAbs, o meio comercial SFM livre de SFB proporcionou melhor resultados para o cultivo do hibridoma PC-61, permitindo que o cultivo em escala laboratorial atingisse densidades celulares ainda maiores que no meio padrão com adição de SFB. Como consequência desse vasto crescimento celular em quantidades abundantes de mAbs foram conseguidas para iniciar num futuro próximo ensaios em modelos animais.