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Antigenic Characterization of <I>Haemophilus somnus</I> LipooligosaccharideHoward, Michael D. 16 November 1998 (has links)
Lipooligosaccharide (LOS) is the major outer membrane component of many Gram-negative bacteria inhabiting the mucosal membranes, including pathogenic species of <I>Haemophilus</I> and <I>Neisseria</I>. LOS phase variation is one mechanism by which some of these bacteria avoid the host immune response. To better understand LOS phase variation as a virulence mechanism of <I>H. somnus</I>, knowledge of the antigenic diversity of LOS epitopes must be increased. Monoclonal antibodies (MAbs) to <I>H. somnus</I> LOS were produced and used with cross-reacting MAbs to <I>H. aegyptius</I> LOS (MAb 5F5) and <I>Neisseria</I> <I>gonorrhoeae</I> LOS (MAb 3F11) in an ELISA to investigate LOS heterogeneity among forty-five strains of <I>H. somnus</I>. Using three MAbs, thirty-nine of these <I>H. somnus</I> strains were grouped into six antigenic types. Three groups, associated solely with the cross-reacting MAbs 5F5 and 3F11, included the majority (76%) of <I>H. somnus</I> strains. The anti-<I>H. somnus</I> LOS MAb 5D7 recognized a low frequency epitope associated with each of the remaining three groups, which included 11% of the <I>H. somnus</I> strains. Six strains (13%) were not recognized by any of these MAbs.
Inhibition ELISA experiments showed that the MAb 5F5 epitope contained phosphocholine (PCho) and this epitope was present in 56% of the strains tested. The MAb 5F5 epitope is phase variable in <I>H. somnus</I> LOS. How PCho negative variants could allow for systemic infection after initial colonization of the mucosa by PCho positive variants is discussed. / Master of Science
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Molecular aspects of antibody mediated T cell activationMorgan, Sara Hannah January 2009 (has links)
The normal physiological activation of naive T cells requires the engagement of both the T cell receptor (TCR) and the co-stimulatory molecule, CD28. However, a group of monoclonal antibodies (mAbs) have been identified that are able to activate T cells in vitro and in vivo via CD28 engagement alone. Two defining characteristics found in all CD28 superagonist mAbs are their membrane proximal CD28 epitopes and the requirement for mAb immobilisation. To investigate whether agonistic mAbs to similar cell molecules could be identified based on epitope position alone, mAbs to the inhibitory receptor PD-1 were generated and characterised. Using a drastic mutation-based epitope mapping technique, one mAb was identified with a membrane proximal epitope along with two other mAbs with membrane distal epitopes. These mAbs were tested for triggering activity in a hybridoma stimulation assay. mAb stimulation was observed with all three mAbs but only in cells expressing a PD-1 chimera that associated with the TCR and the strength of activation was dependent on epitope location. Cross-linking of a monomeric PD-1/CD28 chimera with a pair of anti-PD-1 mAbs resulted in signalling in this system, however, suggesting a role for ligand aggregation in addition to epitope position in mAb signalling. To further investigate the role of epitope position in CD28 superagonism, a cell line expressing a chimeric form of CD28 was created wherein the superagonistic mAb epitope was moved to a membrane distal position. When stimulated with a CD28 superagonist mAb signalling was no longer observed. However stimulation with another mAb that had an epitope to a membrane proximal location on the chimera resulted in superagonistic effect. These results show that epitope location is the dominant cause of T cell stimulation observed by CD28 superagonist mAbs and that epitope dependent mAb signalling is possible in other T cell surface molecules. The work described in this thesis has implications for both the development of immune modulating mAb therapeutics and for the general mechanism of triggering of cell surface receptors dependent on extrinsic tyrosine kinases.
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THE ROLE OF SPIDERS IN THE DETRITAL FOOD WEB OF AN EASTERN DECIDUOUS FORESTHladilek, Erin Elizabeth 01 January 2008 (has links)
Historically, terrestrial food web research has focused on describing the structure of aboveground grazing webs, and determining how interactions among plants, herbivores and higher trophic levels influence primary productivity. Detrital food webs however, play a significant role in regulation of ecosystem dynamics through direct impacts on decomposition. Unraveling the complex nature of detrital food web structure is critical to developing a better understanding of ecosystem function. Therefore the primary objective of this research was to describe the structure of the leaf-litter food web in a temperate deciduous forest, with emphasis on interactions between a community of generalist predators, the forest-floor spiders, and arthropod prey.
Elucidating occurrence of trophic interactions in the forest-floor food web was a formidable task due to the high diversity, small body sizes and cryptic habits of many litter-dwelling arthropods. Analysis of natural variation in consumer stable isotope ratios (δ13C and δ15N) formed the crux of this research because it simultaneously permitted quantification of the trophic positions of litterdwelling arthropods and identification of spider resources, including prey subsidies from the grazing web. A monoclonal antibody-based ELISA was employed to analyze the gut contents of spiders to quantify predation on a major arthropod taxon, the forest-floor flies. Surveys of spider distributions and prey availability in the litter layer also provided fundamental knowledge of community structure.
Stable isotope analyses suggested that most spiders exhibited strong trophic connections to the detrital web, but weak links to herbivorous prey. Several lines of evidence supported a strong trophic link between large, litterdwelling collembolans (Tomoceridae) and cursorial spiders, including correlation between spider and tomocerid densities on the forest-floor, similarities in spider and tomocerid carbon signatures, and nitrogen enrichment of tomocerids relative to other prey types. Conversely, this research provided conflicting evidence regarding spider consumption of flies. Gut content assays indicated consistent predation on flies by cursorial spiders, while stable isotope models suggested that flies are likely of little importance in the spiders’ diets. This project yielded valuable insights into the role of spiders in the forest-floor food web and the potential importance of species-specific variation in prey consumption for detrital food web dynamics.
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Studies of α-synuclein Oligomers-with Relevance to Lewy Body DisordersFagerqvist, Therese January 2013 (has links)
The protein alpha-synuclein (α-synuclein) accumulates in the brain in disorders such as Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). It is believed that the monomeric form of α-synuclein can adopt a partially folded structure and start to aggregate and form intermediately sized oligomers or protofibrils. The aggregation process can continue with the formation of insoluble fibrils, which are deposited as Lewy bodies. The oligomers/protofibrils have been shown to be toxic to neurons and are therefore believed to be involved in the pathogenesis of the actual diseases. The overall aims of this thesis were to investigate the properties of α-synuclein oligomers and to generate and characterize antibodies against these species. In addition, the potential for immunotherapy of the α-synuclein oligomer-selective antibodies were evaluated in a transgenic mouse model with α-synuclein pathology. Stable, β-sheet rich α-synuclein oligomers were induced by incubation with either one of the reactive aldehydes 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE). The oligomers exhibited distinct morphological properties, although both types were toxic when added to a neuroblastoma cell line. The seeding effects of ONE-induced oligomers were studied in vitro and in vivo. The oligomers induced seeding of monomeric α-synuclein in a fibrillization assay but not in a cell model or when injected intracerebrally in transgenic mice. It seemed, however, as if the oligomers affected α-synuclein turnover in the cell model. By immunizing mice with HNE-induced oligomers antibody producing hybridomas were generated. Three monoclonal antibodies were found to have strong selectivity for α-synuclein oligomers. These antibodies recognized Lewy body pathology in brains from patients with PD and DLB as well as inclusions in the brain from young α-synuclein transgenic mice, but did not bind to other amyloidogenic proteins. Finally, immunotherapy with one of the oligomer/protofibril selective antibodies resulted in lower levels of such α-synuclein species in the spinal cord of α-synuclein transgenic mice. To conclude, this thesis has focused on characterizing properties of α-synuclein oligomers. In particular, antibodies selectively targeting such neurotoxic forms were generated and evaluated for passive immunization in a transgenic mouse model. Such immunotherapy may represent a future treatment strategy against Lewy body disorders.
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Modulation des récepteurs de l'adénosine par anticorps monoclonaux et ligands synthétiques. : application en physiopathologie humaine / Modulation of adenosine receptors by monoclonal antibody and synthetized ligands : application in human physiopatologyBy, Youlet 12 November 2010 (has links)
L’adénosine est un nucléoside ubiquitaire qui exerce un contrôle puissant sur les systèmes nerveux,immunitaire et cardiovasculaire par l’intermédiaire de quatre récepteurs membranaires : A1R, A2AR, A2BR etA3R. L’étude des récepteurs de l’adénosine est nécessaire à la compréhension de physio‐pathologieshumaines non encore élucidées. Pour étudier l’expression des A2AR, nous avons, dans une première étude,produit un anticorps monoclonal, appelé Adonis, d’isotype IgM, . Adonis reconnait un épitope linéaire desept acides aminés sur la partie C‐terminale de la seconde boucle extra‐cellulaire de l’A2AR humain. Adonisrévèle, par Western blotting sur lysats cellulaires, une bande de 45 KDa, correspondant à l’A2AR. Adonis secomporte comme un « agonist‐like » en augmentant la production d’AMPc et en inhibant la proliférationcellulaire via la stimulation des A2AR. Dans une deuxième étude, nous avons utilisé Adonis pour montrerque l’expression des A2AR de cellules mononucléées, qui mime celle des tissus cardiaques, permet dedifférencier certains patients souffrant de syncope neurocardiogénique. Nous avons monté dans unetroisième étude, qu’Adonis induit une « down‐régulation » de l’expression des co‐récepteurs CXCR4 etCCR5 des cellules T via la stimulation des A2AR, et qu’à ce titre il pouvait être un outil thérapeutique dans lesinfections par HIV. Dans une quatrième étude, nous avons évalué les effets anti‐nociceptifs d’Adonis qui,administré par voie intra‐cérébro‐ventriculaire, augmente de manière dose‐dépendante les latencesobtenues avec le test du Hot‐plate et du Tail‐flick chez la souris. Ces effets sont renversés par deuxantagonistes des A2AR mais aussi par un antagoniste des récepteurs aux opioïdes. Ceci suggère que leseffets anti‐nociceptifs d’Adonis sont médiés par la libération d’opioïdes endogènes. En marge de sesétudes, nous avons également testé les propriétés biologiques de nouveaux ligands des A1R dans le cadred’une collaboration entre chimistes et biologistes. Ainsi, nous montrons, dans une cinquième étude, queparmi la trentaine de molécules synthétisées, quatre sont des antagonistes et deux autres des agonistesavec un EC50 de l’ordre du micromolaire pour la production d’AMPc. De tels agonistes des A1R pourraientêtre utiles dans le traitement des douleurs neuropathiques, tandis que les antagonistes le seraient dansl’insuffisance cardiaque ou utilisés comme diurétique. Enfin dans une sixième étude, nous avons testé unemolécule originale, puisque bivalente, possédant un pôle d’activité pour les récepteurs aux opioïdes μ et unautre pour les A1R. Cette molécule est un antagoniste pour les deux récepteurs. Elle pourrait avoir desapplications cliniques dans certaines pathologies comme le choc hypovolémique ou le sevrage aux opiacés. / Adenosine interacts on its cell surface receptors, namely A1R, A2AR, A2BR and A3R, to exertphysiological effects on target tissues. Modulation of these adenosine receptors appears to be a currenttopic of research which may bring more comprehensions on human pathophysiology yet to be elucidated.In order to study A2AR expression, we produced, in study 1, a monoclonal antibody anti‐human A2AR, calledAdonis being of IgM, isotype. Adonis recognized a linear epitope of seven amino acids on the C‐terminalpart of the A2AR second extra‐cellular loop. By Western blotting, Adonis reveals a 45 KDa band of A2AR incell lysates. Adonis behaves as an agonist‐like which increases the cAMP production and inhibits cellproliferation through A2AR stimulation. In study 2, we showed that using Adonis, to measure the A2ARexpression of peripheral blood mononuclear cells which mimic those of the cardiac tissue, was able todifferentiate some patients with suspected neurally mediated syncope. We showed, in study 3, that A2ARstimulation by Adonis leads to a down‐regulation of CXCR4 and CCR5 expression on T‐cells, suggesting thatAdonis would be a potential drug to treat HIV infections. In study 4, we showed that intracereboventricularinjection of Adonis increased the Hot‐plate and Tail‐flick test latencies in mice in a dose‐dependent manner.Such increases were prevented by two A2AR antagonists and by an opiate receptor antagonist, suggestingthat the anti‐nociceptive effects of Adonis were mediated, at least in part, by endogenous opioid liberation.The last section focused on biological evaluation of new A1R ligands in collaborative studies betweenchemists and biologists. Indeed we showed, in study 5, that among thirty synthesized molecules, four act asA1R antagonists and two turn out to be A1R agonists with a micromolar EC50 on cAMP production. ThoseA1R agonists would be used in neuropathic pains, whereas other antagonists could be used in cardiacfailure or as diuretic. Finally, in study 6, we tested an original hybrid molecule which was revealed to be abivalent antagonist to μ opiate receptors and A1R. This hybrid compound may have applications in somepathologies such as hypovolemic shock and opiate addiction.
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Comprehensive Glycoproteomics and Glycomics Study of N-Linked Glycans and N-GlycoproteinsLi, Xu 06 January 2017 (has links)
N-linked glycosylation is the most common post-translational modification (PTM) of proteins that exist in nature. N-glycosylation and change in cells serve as a criterion to monitor the activity of developmental stages and diseases severity. Currently, there is an increasing application of mass spectrometry on glycoprotein for malicious, chronic or acute diseases, such as cancers, rheumatoid arthritis (RA) or influenza.
In this dissertation, several mass spectrometric assays have been utilized to, quantitatively and qualitatively, characterize protein N-glycosylation at the glycan, glycopeptide and peptide levels. The goals are to identify serum-based RA biomarker (Chapter 2), or to determine possible glycan structures from monoclonal antibody (Chapter 3), or comprehensively to study one influenza glycoprotein, hemagglutinin (Chapter 4).
In Chapter 2, LC-MS/MS with CID as MS 2 is the primary technique that is applied to collect raw data for RA biomarker screening; western blot is the verification method for newfound biomarkers. This mass spectrometry based comparative analysis of N-glycoprotein in RA and healthy patients’ sera reveal 41 potential biomarkers for RA that can be applied in clinical research. Chapter 3 describes another LC-MS/MS based method developed for the structural analysis of N-glycan released from the monoclonal antibody, immunoglobin G. Higher-energy collision dissociation (HCD) was the surprior technique utilized to identify glycopeptide fragments. The results show that 19 and 23 N-glycan structures were determined from standard and modified mAb samples respectively by using SimGlycan software, while 38 and 35 glycan structures were recognized by manually mapping respectively. 13 N-glycoforms, out of 26 overlapped glycan structures, were identified with significant alterations by comparing standard sample (sample A) and modified mAb (sample B) utilizing our method. In Chapter 4, we comprehensively studied hemagglutinin by using LC-MS/MS and MALDI from both proteomic perspective and glycomics prospective. After confirmed and verified protein sequence and glycosylation sites, galactose-specific quantitation was performed with exoglycosidase digestion combined HPLC with fluorescence detection. The MALDI-MS/MS based method was utilized to confirm glycan structures.
The results in this dissertation provide insights into the significance of protein glycosylation alterations as RA biomarkers, and these quantitative methods can be reapplied to any other disease biomarkers screening for clinical researchers.
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B cell and antibody responses to influenza A virus in humanHuang, Kuan-Ying January 2011 (has links)
Neutralising antibodies and antigen-specific B cells are important for protection against influenza A virus. However, the antigenic evolution of influenza A viruses has made a continuing challenge to the design of vaccine and the public health. The ability to generate cross-reactive response against influenza remains unclear in human. It is important to explore the antibody and B cell repertoire at single cell level. The pandemic H1N1 and seasonal influenza vaccine induced robust antibody response in adults. However, pre- or co-vaccination with the seasonal vaccine led to a significantly reduced antibody response to pandemic H1N1 virus. Whether this interference has impact on subsequent infection rates remains undetermined. There observed substantial cross-reactive antibody response upon vaccination, as measured by HI, MN and B cell ELISpot assays. The antibody recognizing conserved proteins could be the main component of cross-reactivity against influenza A strains and subtypes. A significant expansion of influenza-specific MBC was observed after infection. Crossreactive response was also noted in the MBC response. Importantly, a robust early-phase ASC response was detected in the peripheral blood upon influenza vaccination or infection. The size of ASC response significantly correlated with serum HI, MN and anti-HA IgG titre three weeks after vaccination. The sequence analysis revealed that early-phase ASC accumulated high level of somatic mutations on Ig variable region and affinity maturation, as well as anti-influenza mAb, which suggested their origin from pre-existing MBC. Eight anti-influenza mAb were made from early-phase ASC, including one high-titre virus-neutralising HA1-specific, two other HA1-specific, one cross-reactive HA2-specific, and four cross-reactive NP-specific antibodies, indicating of the broad diversity of ASC repertoire. In conclusion, this study demonstrated the properties of antibody and B cell responses to influenza A virus at serological, cellular and sequence level. The virus-neutralising and cross-reactive mAb derived from ASC could have therapeutic potential and their analysis might direct the vaccine design in the near future.
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"Estudo de marcação com Iodo-131 de anticorpo monoclonal anti-CD20 usado na terapia de linfoma não-Hodgkin" / The study of labeling with iodine-131 of monoclonal antibody anti-CD20 used for the treatment of non-Hodgkin lymphomaAkanji, Akinkunmi Ganiyu 01 December 2006 (has links)
Linfomas são doenças originárias do sistema linfático, descritos por Thomas Hodgkin em 1932. São tradicionalmente classificados em dois grupos básicos: doenças de Hodgkin e linfomas do tipo não hodgkin (LNH). Inicialmente, pacientes com LNH foram tratados com radioterapia apenas ou combinada com imunoterapia usando-se anticorpo monoclonal anti-CD20 (ex., Rituximab-Mabthera, Roche). Porém, radioimunoterapia é uma nova modalidade de tratamento para pacientes portadores de LNH, na qual radiação citotóxica proveniente de radioisótopos terapêuticos é depositada nos tumores via anticorpos monoclonais (Acms). Este estudo concentrou-se nas condições de marcação do Acm anti-CD20 (Rituximab-Mabthera, Roche), com radioiodo (131I), pelo método direto, usando-se Cloramina-T como agente oxidante. Foram estudados parâmetros de marcação tais como: método de purificação, influência de tempo de incubação, influência de massa de agente oxidante, estabilidade in vitro e in vivo, imunoreatividade do anticorpo e distribuição biológica do anticorpo marcado em camundongos Swiss sadios. Produto de alta pureza radioquímica foi obtido, sem diferença notável entre os métodos aplicados. Não foi observada nenhuma influência direta do tempo de incubação na pureza radioquímica do anticorpo marcado. Um pequeno decréscimo na pureza radioquímica foi observado quando variou-se a massa do Acm sem variar a atividade do radioiodo. Após purificação, o anticorpo marcado apresentou pureza radioquímica de aproximadamente 100 %. Observou-se um produto de alta pureza radioquímica quando o anticorpo foi marcado na condição padrão. O anticorpo anti-CD20 apresentou variações de pureza radioquímica quando sua estabilidade foi testada em cinco condições estabilizadoras diferentes. Entretanto, a condição em que ácido gentísico foi combinado com congelamento demonstrou-se apropriada e capaz de minimizar os efeitos de autoradiólise do anticorpo marcado com alta atividade terapêutica de iodo-131. O anticorpo marcado apresentou imunoreatividade abaixo da relatada na literatura. A distribuição biológica em camundongos Swiss sadios revelou captação elevada no pulmão, fígado, e intestino delgado. O clareamento sanguíneo do anticorpo marcado foi relativamente rápido. Contudo, dados experimentais revelaram que anticorpos monoclonais anti-CD20 podem ser seguramente marcados com alta atividade de iodo-131 usando o método de Cloramina-T. O uso de cromatografia em camada delgada (ITLC-SG) na avaliação de pureza radioquímica mostrou-se apropriado, eficiente, prático, além de simples e rápido. O método de purificação utilizado demonstrou-se eficiente para a separação do anticorpo marcado do iodo livre. A abordagem inédita apresentada neste estudo, no sentido de viabilizar transporte e comercialização do produto marcado, referiu-se ao estudo de estabilidade do Acm marcado. A distribuição biológica do anticorpo demonstrou-se compatível com a distribuição do anticorpo íntegro indicando ótima estabilidade relativa in vivo. Os resultados deste estudo confirmam a potencialidade do anticorpo para a radioimunoterapia de linfomas do tipo não-Hodgkin. / Lymphomas are malignancies of the lymphatic system, described by Thomas Hodgkin in 1932. Traditionally, lymphomas are classified in two basic groups: Hodgkin disease and non-Hodgkin lymphoma (NHL). Patients with NHL were earlier treated with radiotherapy alone or in combination with immunotherapy using monoclonal antibody anti-CD20 (ex., Rituximab-Mabthera, Roche). However, Radioimmunotherapy is a new modality of treatment for patients with NHL, in which cytotoxic radiation from therapeutic radioisotopes is delivered to tumors through monoclonal antibodies. This study focused on labeling conditions of monoclonal antibody anti-CD20 (Rituximab-Mabthera, Roche) with iodine-131, by direct radioiodination method using Chloramine-T as oxidizing agent. Labeling parameters investigated were: Radiochemical purity (RP), method of purification, incubation time, antibody mass, oxidative agent mass, stability in vitro, stability in vivo, immunoreactivity and biological distribution performed in normal Swiss mouse. Product of high radiochemical purity was obtained with no notable difference between the methods applied. No clear evidence of direct influence of incubation time on radiochemical purity of the labeled antibody was observed. Whereas, a clear evidence of direct influence of activity on radiochemical purity of the labeled antibody was observed when antibody mass was varied. After purification, the labeled product presented radiochemical purity of approximately 100 %. Product of superior radiochemical yield was observed when standard condition of labeling was used. The labeled product presented variation in radiochemical purity using five different stabilizer conditions. The condition in which gentisic acid was combined with freeze appears more suitable and capable of minimizing autoradiolysis of the antibody labeled with high therapeutic activity of iodine-131. The labeled product presented low immunoreactivity when compared to the literature. Biological distribution in normal Swiss mouse demonstrated high uptake of the labeled antibody in lungs, liver, and small intestine. The progressive loss of activity in blood indicates fast blood clearance of the labeled antibody that is eliminated through the kidney, in urine. The experimental data proved that mAb anti-CD20 can be securely labeled with high therapeutic activity of iodine-131 using Chloramine-T method. Radiochemical purity determined by chromatographic plates (ITLC-SG) proved to be appropriate, efficient, practical and simple. The purification method demonstrated to be appropriate and efficient for separating the labeled antibody from free iodine. The results of stability of the labeled antibody presented in this study suggest that the product can be transported and commercialized using the condition in which gentisic acid was combined with freeze. In vivo distribution of the labeled antibody shows to be compatible with integral antibody distribution, indicating good in vivo stability. Results obtained in this study confirmed the potential of the labeled product anti-CD20-131I for radioimmunotherapy of non-Hodgkin lymphoma (NHL).
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"Estudo de marcação com Iodo-131 de anticorpo monoclonal anti-CD20 usado na terapia de linfoma não-Hodgkin" / The study of labeling with iodine-131 of monoclonal antibody anti-CD20 used for the treatment of non-Hodgkin lymphomaAkinkunmi Ganiyu Akanji 01 December 2006 (has links)
Linfomas são doenças originárias do sistema linfático, descritos por Thomas Hodgkin em 1932. São tradicionalmente classificados em dois grupos básicos: doenças de Hodgkin e linfomas do tipo não hodgkin (LNH). Inicialmente, pacientes com LNH foram tratados com radioterapia apenas ou combinada com imunoterapia usando-se anticorpo monoclonal anti-CD20 (ex., Rituximab-Mabthera, Roche). Porém, radioimunoterapia é uma nova modalidade de tratamento para pacientes portadores de LNH, na qual radiação citotóxica proveniente de radioisótopos terapêuticos é depositada nos tumores via anticorpos monoclonais (Acms). Este estudo concentrou-se nas condições de marcação do Acm anti-CD20 (Rituximab-Mabthera, Roche), com radioiodo (131I), pelo método direto, usando-se Cloramina-T como agente oxidante. Foram estudados parâmetros de marcação tais como: método de purificação, influência de tempo de incubação, influência de massa de agente oxidante, estabilidade in vitro e in vivo, imunoreatividade do anticorpo e distribuição biológica do anticorpo marcado em camundongos Swiss sadios. Produto de alta pureza radioquímica foi obtido, sem diferença notável entre os métodos aplicados. Não foi observada nenhuma influência direta do tempo de incubação na pureza radioquímica do anticorpo marcado. Um pequeno decréscimo na pureza radioquímica foi observado quando variou-se a massa do Acm sem variar a atividade do radioiodo. Após purificação, o anticorpo marcado apresentou pureza radioquímica de aproximadamente 100 %. Observou-se um produto de alta pureza radioquímica quando o anticorpo foi marcado na condição padrão. O anticorpo anti-CD20 apresentou variações de pureza radioquímica quando sua estabilidade foi testada em cinco condições estabilizadoras diferentes. Entretanto, a condição em que ácido gentísico foi combinado com congelamento demonstrou-se apropriada e capaz de minimizar os efeitos de autoradiólise do anticorpo marcado com alta atividade terapêutica de iodo-131. O anticorpo marcado apresentou imunoreatividade abaixo da relatada na literatura. A distribuição biológica em camundongos Swiss sadios revelou captação elevada no pulmão, fígado, e intestino delgado. O clareamento sanguíneo do anticorpo marcado foi relativamente rápido. Contudo, dados experimentais revelaram que anticorpos monoclonais anti-CD20 podem ser seguramente marcados com alta atividade de iodo-131 usando o método de Cloramina-T. O uso de cromatografia em camada delgada (ITLC-SG) na avaliação de pureza radioquímica mostrou-se apropriado, eficiente, prático, além de simples e rápido. O método de purificação utilizado demonstrou-se eficiente para a separação do anticorpo marcado do iodo livre. A abordagem inédita apresentada neste estudo, no sentido de viabilizar transporte e comercialização do produto marcado, referiu-se ao estudo de estabilidade do Acm marcado. A distribuição biológica do anticorpo demonstrou-se compatível com a distribuição do anticorpo íntegro indicando ótima estabilidade relativa in vivo. Os resultados deste estudo confirmam a potencialidade do anticorpo para a radioimunoterapia de linfomas do tipo não-Hodgkin. / Lymphomas are malignancies of the lymphatic system, described by Thomas Hodgkin in 1932. Traditionally, lymphomas are classified in two basic groups: Hodgkin disease and non-Hodgkin lymphoma (NHL). Patients with NHL were earlier treated with radiotherapy alone or in combination with immunotherapy using monoclonal antibody anti-CD20 (ex., Rituximab-Mabthera, Roche). However, Radioimmunotherapy is a new modality of treatment for patients with NHL, in which cytotoxic radiation from therapeutic radioisotopes is delivered to tumors through monoclonal antibodies. This study focused on labeling conditions of monoclonal antibody anti-CD20 (Rituximab-Mabthera, Roche) with iodine-131, by direct radioiodination method using Chloramine-T as oxidizing agent. Labeling parameters investigated were: Radiochemical purity (RP), method of purification, incubation time, antibody mass, oxidative agent mass, stability in vitro, stability in vivo, immunoreactivity and biological distribution performed in normal Swiss mouse. Product of high radiochemical purity was obtained with no notable difference between the methods applied. No clear evidence of direct influence of incubation time on radiochemical purity of the labeled antibody was observed. Whereas, a clear evidence of direct influence of activity on radiochemical purity of the labeled antibody was observed when antibody mass was varied. After purification, the labeled product presented radiochemical purity of approximately 100 %. Product of superior radiochemical yield was observed when standard condition of labeling was used. The labeled product presented variation in radiochemical purity using five different stabilizer conditions. The condition in which gentisic acid was combined with freeze appears more suitable and capable of minimizing autoradiolysis of the antibody labeled with high therapeutic activity of iodine-131. The labeled product presented low immunoreactivity when compared to the literature. Biological distribution in normal Swiss mouse demonstrated high uptake of the labeled antibody in lungs, liver, and small intestine. The progressive loss of activity in blood indicates fast blood clearance of the labeled antibody that is eliminated through the kidney, in urine. The experimental data proved that mAb anti-CD20 can be securely labeled with high therapeutic activity of iodine-131 using Chloramine-T method. Radiochemical purity determined by chromatographic plates (ITLC-SG) proved to be appropriate, efficient, practical and simple. The purification method demonstrated to be appropriate and efficient for separating the labeled antibody from free iodine. The results of stability of the labeled antibody presented in this study suggest that the product can be transported and commercialized using the condition in which gentisic acid was combined with freeze. In vivo distribution of the labeled antibody shows to be compatible with integral antibody distribution, indicating good in vivo stability. Results obtained in this study confirmed the potential of the labeled product anti-CD20-131I for radioimmunotherapy of non-Hodgkin lymphoma (NHL).
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Estudo de marcação do anticorpo monoclonal anti-PBP2a com 99mTc / The labelling of antibody anti-PBP2a with 99mTcMororó, Janio da Silva 04 September 2012 (has links)
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. / Staphylococcus aureus is a major cause of life-threatening infections such as bacteremia and endocarditis. Unfortunately, many strains of this bacterial species have become resistant to certain antibiotics, including methicillin and amoxicillin. These strains are known as methicillin-resistant S. aureus (MRSA). The penicillin binding protein 2a (PBP2a) is the enzyme responsible for conferring resistance β-lactams antibiotics for MRSA, being one promising molecule for therapy with mAb. However, besides the therapy, the methods of diagnosis are also inefficient because the diagnosis currently takes several days to produce a reliable result. Taking into account, the objective of this research was radiolabeling one anti-PBP2a mAb developed by Bio-Manguinhos/FioCruz-RJ, utilizing 99mTc, for in situ diagnostic of the infectious caused by MRSA. First, anti-PBP2a mAb was reduced utilizing 2-mecaptoethanol (2-ME) for generate sulphydryl groups (-SH) and after to be labeled with 99mTc. In this work, were utilized two techniques of direct method: Method 1, using tartrate and gentisic acid reagents, acting like transchelant and stabilizer agents, respectively; and Method 2, using one commercial kit of MDP. Besides the radiolabeling, the mAb reduced and mAb labeled with 99mTc were submitted to immunoreactivity analysis, with SDS-PAGE non-reducing, Immunoblotting, ELISA and neutralization assay in vitro methods. The quantity produced of sulphydryl groups by mAb was satisfactory, approximately 5 per mAb, utilizing 6.500:1 of 2-ME:mAb molar ratio. The better labeling method was Method 2, with labeling yield of 73.5%, and showed a good stability after 2 hours (73.2%). The better formulation was: 0.5 mg of mAb anti- PBP2a, 10 μL of MDP kit, after resuspended with 5 mL of saline, and 75.48 MBq (2.04 mCi) of 99mTc, reacting by 15 minutes. The labeled mAb maintained the immunoreactivity, utilizing immunologic and in vitro experiments.
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