Made available in DSpace on 2014-10-09T12:33:45Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:03:58Z (GMT). No. of bitstreams: 0 / Dissertação (Mestrado) / IPEN/D / Instituto de Pesquisas Energéticas e Nucleares - IPEN-CNEN/SP

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Made available in DSpace on 2014-10-09T12:35:29Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:04:36Z (GMT). No. of bitstreams: 0 / Staphylococcus aureus é um dos principais microorganismos causadores de infecção em humanos, podendo causar inclusive bacteremia e endocardite nos indivíduos infectados. Diversas cepas desta bactéria apresentam resistência a diferentes tipos de antibióticos, dentre eles os antibióticos meticilina e amoxicilina, como no caso da bactéria Staphylococcus aureus resistente à meticilina (MRSA). A Proteína ligadora de Penicilina 2a (PBP2a) é a enzima responsável por conferir resistência para a MRSA aos antibióticos β-lactâmicos, sendo uma molécula promissora para terapia com AcM. No entanto, além das terapias os métodos de diagnóstico também são ineficientes, pois atualmente o diagnóstico leva vários dias para produzir um resultado confiável. O objetivo deste trabalho foi desenvolver um radiofármaco utilizando o AcM anti-PBP2a, desenvolvido em Bio-Manguinhos/FioCruz, marcado com 99mTc, para identificação in situ do foco infeccioso causado por MRSA. Neste trabalho, incialmente o AcM anti-PBP2a foi reduzido com o agente redutor 2-mercaptoetanol (2-ME), para gerar grupos sulfidrilas (- SH). Logo após foram utilizados dois diferentes métodos da Marcação Direta: o Método 1, utilizando o reagente tartarato e o ácido gentísico, que atuam como agente transquelante e estabilizador, respectivamente; e o Método 2, utilizando um kit comercial de MDP, no qual o MDP atua como agente transquelante. Após a marcação do anticorpo, o radiofármaco foi submetido a ensaios de avaliação funcional, utilizando os métodos de eletroforese em gel SDS-PAGE não redutor; Immunoblotting; ELISA e o Ensaio de Neutralização in vitro. Como resultado foi visto que a quantidade média de grupos sulfidrilas produzida por AcM foi considerada satisfatória, cerca de 5 grupos SH por IgG, utilizando para isto a relação molar de 6.500:1 de 2-ME:AcM. O Método 2 foi o método que obteve melhor rendimento de marcação, com 73,5%, apresentando boa estabilidade depois de 2 horas (73,2%). A melhor formulação utilizada foi a seguinte: 0,5 mg de AcM anti-PBP2a; 10 μL do kit do MDP, depois de ser resuspendido com 5 mL de solução salina; e 75,48 MBq (2,04 mCi) de 99mTc, a reação ocorrendo em 15 minutos. Os Ensaios de Avaliação Funcional demonstraram que o AcM manteve a especificidade e afinidade de ligação à PBP2a. / Dissertação (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Protein A (ProA) chromatography is a bioseparations technique employed throughout the biopharmaceutical industry for the selective capture and purification of IgG-class monoclonal antibodies (mAbs) and Fc-fusion proteins. The rapid growth of mAbs as commercial therapeutics has motivated the need for improved, efficient, and high-throughput purification processes during manufacturing. In direct response, the work presented in thesis aims to 1) increase the scientific community’s understanding of IgG adsorption behavior on ProA chromatography resins and 2) improve the performance of ProA chromatography with ligands that are chemically modified using polyethylene glycol (PEGylated). The results of this thesis suggest that IgG molecules of varying binding strength, or varying elution pH, are capable of competing for binding sites on ProA chromatography resins in simultaneous or sequential adsorption. The competitive phenomenon derives from variance in IgG binding strength, or IgG elution pH, due to differences in sub-class behavior as well as secondary IgG binding interactions with the ProA ligand. Competition is readily apparent in the adsorption of human polyclonal IgG, which has a wide variety of IgG sub-classes and binding epitopes. Additionally, the results presented in this thesis suggest that ProA chromatography resins with PEGylated ligands are a viable path to increase resin robustness and real-world chromatographic selectivity. It is demonstrated that ligand PEGylation can increase resistance to proteolytic digestion, mitigate impurity interactions with mAbs that are bound to ProA, and increase process selectivity against Chinese Hamster Ovary host cell proteins by up to 37%. However, resins with large volumes of conjugated PEG significantly decrease IgG static binding capacity and decrease the available pore space for diffusion, resulting in losses in dynamic binding capacity and productivity. Lighter modifications appear to avoid losses in dynamic binding capacity, however, they do not appear to be effective at mitigating impurity interactions with mAbs that are bound to ProA, which is key to increasing process selectivity. PEGylation of ProA also universally increases the elution pH of IgG molecules by weakening the binding interaction. This last result opens another path of viability for PEGylated ProA ligands for purification of mAbs of Fc-fusion proteins that are sensitive to low pH environments.

Six mouse monoclonal antibodies have been isolated which react against a bacterial fusion protein containing amino acids 364 to 623 of the NS-1 protein of the prototype strain of the Minute Virus of Mice (MVMp). All six were found to be of the IgG class of antibodies; five being IgG₁ and the sixth being IgG₂[formula omitted]. By immunoblot analyses, these antibodies all recognize an 83 kDa protein found only in MVM-infected mouse fibroblast cells, leading to the assumption that they are all NS-1 specific. Further evidence for this assumption is obtained from indirect immunofluorescence studies showing all but one of the mAbs react against a nuclear protein found in MVM-infected cells. The epitopes of the antibodies were mapped using carboxy-terminal deleted bacterial fusion proteins derived from the plasmid encoding the original antigen. For the six monoclonal antibodies, four distinct epitopes were found (A - D). Three were clustered in a 16 amino acid region near the carboxy-terminal of the bacterial fusion protein, while the fourth was slightly more toward the amino-terminal side. Competition ELISAs against a 25 amino acid NS-1 specific peptide confirmed the mapping of the A epitope recognized by the CE10 and AC6 monoclonal antibodies. Also in this thesis, the characterization of a NS-1 fusion protein and a non-fused NS-1 protein expressed in insect cells by recombinant baculoviruses is also described. The latter, a full-length NS-1 protein designated NS-1[formula omitted]ⅽ, was found to be an 84 kDa cytoplasmic protein. This protein was immunoprecipitated by all six monoclonal antibodies. A CE10 monoclonal antibody immunoaffinity column was employed in the single-step purification of NS-1 [formula omitted]c from insect cells. Four elution methods (alkaline, peptide, 6M guanidinium, and acid) were examined and the best purification was obtained using the acid elution. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

No description available.

In this study, the production of monoclonal antibodies directed against the activating enzyme for an S6 kinase is examined and described. Evidence is presented for the association of an Mr. 55,000 abd Mr. 95,000 protein with the s6 kinase. These proteins are phosphorylated in the presence of Activating Enzyme. A sequence of regulatory events for insulin-stimulated phosphorylation of ribosomal protein S6 in cells is postulated as follows: insulin activates the receptor tyrosine kinase, which phosphorylates the Mr 116,000 subunit of Activating Enzyme. The Activating Enzyme then activates the S6 kniase by phosphorylation, and phosphorylation of the ribosomal protein s6 is promoted.

Call number: LD2668 .T4 1984 H35 / Master of Science

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Immunochemical reagents were characterized under carefully controlled laboratory conditions using conventional high performance liquid chromatography instrumentation. The stationary phase was prepared by attaching antigen molecules to an insoluble support through a covalent linkage. Experiments were carried out by introducing antibody molecules into the mobile phase and monitoring their interaction with the stationary phase. Monoclonal antibodies were employed because of their more homogeneous properties compared to polyclonal antisera. Radioisotopes were employed to study low level adsorption on the stationary phase. Recovery experiments were carried out in which it was possible to account for all of the material introduced into the mobile phase. Antibodies were purified over a preparative scale antigen affinity column following labeling to insure high immunoreactivity. Studied under normally dissociating conditions, irreversible adsorption of picomole amounts of protein on the antigen stationary phase was greater than on other ligand modified stationary phases. This accumulation decreased with repeated use of the affinity column. The present study provides a framework for evaluation of other immunoaffinity systems and demonstrates that reproducible recovery of immunologically active material in high yield is possible. Monoclonal antibodies labeled with fluorescein were different from unlabeled molecules in binding and physical characteristics. Computer simulations were used to describe binding behavior. Although fluorescein labels improve detection sensitivity over native protein absorbance, their use in this case decreased binding affinity significantly. Heterogeneity of affinity purified fluorescein labeled and unlabeled monoclonal antibodies was examined with two dimensional gel electrophoresis. In addition to increased charge heterogeneity in the labeled antibody fragments, both light and heavy chains possessed more negative character. These results agree with each other. Fluorescein contains a carboxylic acid group, and modification of antibody light chains may interfere with binding affinity. The number and location of labels covalently attached to antibodies must be carefully controlled to obtain maximum detection sensitivity and preserve immunoreactivity.