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- ItemAcesso aberto (Open Access)Anticorpos monoclonais(Universidade Federal de São Paulo, 2021-02-05) Conceição, Ana Beatriz Alves da [UNIFESP]; Xander, Patricia [UNIFESP]; http://lattes.cnpq.br/3620553457348403; http://lattes.cnpq.br/6687689950877389Anticorpos monoclonais (mAbs) são moléculas de imunoglobulinas idênticas produzidas para um antígeno específico. Anticorpos são estruturalmente compostos por 2 cadeias leves e 2 cadeias pesadas que contém o sítio de ligação ao antígeno e a região que se liga à receptores das células do sistema imunológico. O primeiro anticorpo monoclonal foi produzido em 1975 com a técnica de produção de hibridoma, onde há fusão de uma célula de mieloma com uma célula B de um animal previamente imunizado. Porém, anticorpos murinos apresentam diferenças em relação aos anticorpos humanos e, por isso, foram relatadas reações de hipersensibilidade e rápida eliminação do organismo, comprometendo sua função terapêutica. Desde então, várias técnicas surgiram com o objetivo de aumentar a segurança e eficácia do mAbs. Essas técnicas incluem: anticorpos humanizados e quiméricos, produzidos pela tecnologia de DNA recombinante, por Phage Display, ou animais transgênicos; e anticorpos monoclonais humanos, produzidos por tecnologia de célula B única. Atualmente, há mais de 80 anticorpos monoclonais aprovados para a clínica, sendo a maior parte deles para tratamento de doenças, como câncer. Neste trabalho, foi realizada revisão bibliográfica com o objetivo de identificar e discutir as principais técnicas para obtenção de anticorpos monoclonais e suas aplicações.
- ItemAcesso aberto (Open Access)Captura de anticorpos monoclonais anti-IgG humana a partir de sobrenadante de cultura celular por precipitação com polietilenoglicol e cloreto de zinco(Universidade Federal de São Paulo, 2023-02-24) Pássaro, Ana Carolina Moreno [UNIFESP]; Bresolin, Igor Tadeu Lazzarotto [UNIFESP]; Bresolin, Iara Rocha Antunes Pereira [UNIFESP]; http://lattes.cnpq.br/5925807286464060; http://lattes.cnpq.br/5750111176589237; http://lattes.cnpq.br/1973730368476455Os anticorpos monoclonais (mAbs) são biomoléculas que se tornaram fundamentais em diversas áreas de estudo, sendo capazes de reconhecer analitos em uma mistura complexa e, desta forma, contribuir em dispositivos de detecção e diagnóstico médico, como os testes imunodiagnósticos rápidos. Estes testes utilizam, como um de seus reagentes de captura, anticorpos do tipo anti-IgG humana que atuam no processo de detecção de IgG policlonal no sangue de indivíduos humanos. Neste sentido, este trabalho teve como finalidade estudar a técnica de precipitação com polietilenoglicol (PEG) e ZnCl2 como alternativa não-cromatográfica de captura para mAbs de aplicações não-terapêuticas, porém que ainda requerem alto grau de pureza, tal como os utilizados em testes imunodiagnósticos. Os experimentos foram conduzidos utilizando PEG de diferentes massas moleculares e concentrações (4000 e 6000 g mol-1, variando de 0 a 16% m/v) e ZnCl2 (0 a 16 mmol L-1) como agentes precipitantes para mAbs IgG1 de camundongo do tipo anti-IgG humana provenientes de sobrenadante de cultura celular de hibridomas, determinando-se suas curvas de solubilidade e as condições mais promissoras para precipitação em batelada, além de otimizar a etapa de ressuspensão e realizar uma análise de viscosidade para os anticorpos ressuspensos. A concentração de anticorpos foi avaliada por cromatografia de afinidade analítica com proteína A imobilizada em matriz sólida e em eluição gradiente em HPLC (High Performance Liquid Chromatography), estimando-se a pureza dos precipitados por cromatografia por exclusão de tamanho em eluição isocrática, também em HPLC. Os experimentos resultaram na obtenção de um precipitado com rendimento de 100% e pureza de 15% (obtido por precipitação com PEG6000 14%) e de um precipitado de 100% de rendimento e 59% de pureza (obtido por precipitação com PEG6000 12% combinado com ZnCl2 3 mmol L-1). A análise de viscosidade indicou que a combinação de PEG6000 e ZnCl2 como agentes precipitantes não permitiu uma redução da viscosidade dinâmica do sobrenadante de precipitação (5,96 mPa s) quando comparado ao sobrenadante obtido com apenas PEG6000 (5,71 mPa s), embora tenha resultado em um precipitado com viscosidade menor (2,41 mPa s) que a do sobrenadante. Visto que a pureza máxima obtida foi de cerca de 60%, para que os mAbs se tornem elegíveis para a aplicação mencionada (pureza mínima de 98%), recomenda-se a complementação com outro método de purificação, bem como a possibilidade do uso de pré-operações de concentração do material de partida.
- ItemAcesso aberto (Open Access)Development of a prototype immunochromatographic test for rapid diagnosis of respiratory adenovirus infection(Elsevier Brazil, 2017) Paulini, Inarei [UNIFESP]; Siqueira-Silva, Joselma; Thomaz, Luciana; Rocha, Leticia; Harsi, Charlotte; Bellei, Nancy [UNIFESP]; Granato, Celso [UNIFESP]Human adenoviruses comprise an important group of etiologie agents that are responsible for various diseases in adults and children, such as respiratory, ocular, gastroenteric, and urinary infections. In immunocompromised and organ-transplanted individuals, these agents can cause generalized infections. Rapid diagnostic methods for detecting these infectious agents are not widely available.& para;& para;The aim of this work was to produce monoclonal and polyclonal anti-adenovirus antibodies to be used in a rapid diagnostic test for respiratory infections.& para;& para;Adenovirus hexons were satisfactorily purified by ultracentrifugation and chromatography. After virus purification, anti-hexon monoclonal antibodies were produced and characterized, following classical methods. Antibodies were specific for adenoviruses 2,3,5, and 41. The proposed immunochromatographic test was standardized using colloidal gold.& para;& para;The standardization of the rapid test was sufficient to detect adenovirus antigens (in nasopharyngeal lavage samples) with sensitivity of 100% and specificity of 85% when compared to direct immunofluorescence.& para;& para;The immunochromatographic assay prototype was sufficiently sensitive to detect B (3), C (2 and 5), and F (41) adenovirus samples. Although based on preliminary data, the test demonstrated the same performance as direct immunofluorescence, but with the advantage of being a point-of-care test. Further studies are still needed to confirm its effectiveness in clinical practice. (C) 2017 Sociedade Brasueira de Infectologia. Published by Elsevier Editera Ltda.
- ItemAcesso aberto (Open Access)Hairy cell leukemia: a histo-cytochemical and ultra-structural study(Associação Paulista de Medicina - APM, 1998-03-01) Gonsalez, Denize [UNIFESP]; Oliveira, José Salvador Rodrigues de [UNIFESP]; Freymüller-Haapalainen, Edna [UNIFESP]; Kerbauy, José [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)We studied five patients with hairy cell leukemia (HCL) diagnosed within the last ten years at the Department of Hematology of Universidade Federal de São Paulo (UNIFESP) - Escola Paulista de Medicina. Our purpose was to analyze the value of transmission electron microscopy (TEM) by comparing this method with the conventional ones. At diagnosis, patients presented weight loss, spleen enlargement and hairy cells (HC) in peripheral blood and bone marrow slides. HC was characterized by morphology and tartrate test resistance in the acid phosphatase reaction (TRAP). At the evaluation time, the amount of HC ranged from 1% to 85% of WBC count. All patients, except two, had phenotype B. In these last two, TRAP as well as phenotype B could not be documented due to low HC numbers in their exams. Cytoplasmatic projections and the absence of lamellar ribosomic complex were the most frequent ultrastructural findings, even in those patients with the lowest HC numbers. Based on these features, TEM is an efficient method for searching for HC at HCL diagnosis and during the course of the disease.
- ItemAcesso aberto (Open Access)Identificação, por anticorpo monoclonal, de proteína de 230 kDa relacionada com malignidade em melanoma murino(Universidade Federal de São Paulo (UNIFESP), 2006-12-31) Mendes, Priscila Fraga Penteado [UNIFESP]; Lopes, Jose Daniel [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Melanomas se destacam entre os tumores sólidos por apresentar alto potencial de malignidade e incidência crescente, especialmente entre indivíduos jovens. Identificação de marcadores moleculares em melanomas é de enorme interesse para uso clínico e para estudos relacionados ao seu desenvolvimento. A linhagem de melanoma murino B16 tem sido amplamente empregada visando melhor compreensão do processo metastático. Objetivo: identificar moléculas na superfície de células B16, empregando anticorpos monoclonais, que apresentem função biológica importante para essas células, bem como investigar expressão de moléculas reconhecidas pelos mesmos mAbs em melanoma humano. Métodos: camundongos C57Bl/6 foram imunizados com células B16 irradiadas para produção de híbridos produtores de mAb. Resultados: após fusão foi obtido mAb da classe IgM, designado G12F2, que reconheceu uma única banda de aproximadamente 230 kDa em extrato total de células B16. A molécula era expressa na superfície celular e não por células não tumorigênicas, como fibroblastos ou melanócitos melan-a. Células não tumorigênicas, derivadas de melan-a, também não a expressaram ao passo que células tumorigênicas, de mesma origem, expressaram-na em grande quantidade. Variante menos metastática da linhagem B16 expressou menor quantidade desta molécula quando comparado à variante mais metastática. A neutralização da molécula de 230 kDa com mAb G12F2 inibiu proliferação, migração e invasão por células B16 in vitro. Também nestas condições, G12F2 promoveu atividade citolítica contra células B16, mediada por complemento. Por outro lado, adição in vitro de mAb G12F2 em nada alterou adesão das células B16 à fibronectina e laminina, ou adesão célula-célula. In vivo, o tratamento com mAb G12F2 inibiu crescimento do nódulo tumoral e formação de metástases pulmonares. Quando testado contra extrato de tumores de origem humana, como carcinoma e melanoma, mAb G12F2 reconheceu banda de 75 e 67 kDa, respectivamente. Por fim, foi demonstrado que mAb inibiu proliferação de células de melanoma humano in vitro. Conclusões: a molécula de 230 KDa parece ter importância durante crescimento do melanoma murino; identificação de molécula homóloga em melanoma humano fornecerá subsídios para diagnóstico e protocolos visando imunoterapia.
- ItemSomente MetadadadosIdentification of enolase as a laminin-binding protein on the surface of Staphylococcus aureus(Elsevier B.V., 2004-05-01) Carneiro, Celia Regina Whitaker [UNIFESP]; Postol, Edilberto; Nomizo, Regina; Reis, Luiz Fernando Lima; Brentani, Ricardo Renzo; Hosp Canc AC Camargo; Universidade Federal de São Paulo (UNIFESP); Universidade de São Paulo (USP); Ludwig Inst Canc ResWe have previously demonstrated that Staphylococcus aureus, a highly invasive bacteria, presents a 52-kDa surface protein that mediates its binding to laminin. in order to better characterize this receptor, we excised this putative laminin receptor from two-dimensional (2-D) PAGE and used it as antigen for raising a mouse hyperimmune serum which was for screening an S. aureus expression library. A single clone of 0.3 kb was obtained, and its sequence revealed 100% homology with S. aureus a-enolase. Moreover, amino acid sequencing of the 52-kDa protein eluted from the 2-D gel indicated its molecular homology with a-enolase, an enzyme that presents a high evolutionary conservation among species. in parallel, monoclonal antibodies raised against the S. aureus 52-kDa band also recognized yeast a-enolase in western blot analysis. These monoclonal antibodies were also able to promote capture of iodine-labeled bacteria when adsorbed to a solid phase, and this capture was inhibited by the addition of excess rabbit muscle a-enolase. Finally, the cell surface localization of S. aureus a-enolase was further confirmed by flow cytometry. Hence, a-enolase might play a critical role in the pathogenesis of S. aureus by allowing its adherence to laminin-containing extracellular matrix. (C) 2004 Elsevier SAS. All rights reserved.
- ItemSomente MetadadadosImmunochemical and biological characterization of monoclonal antibodies against BaP1, a metalloproteinase from Bothrops asper snake venom(Elsevier B.V., 2010-11-01) Fernandes, I.; Assumpcao, G. G.; Silveira, C. R. F.; Faquim-Mauro, E. L.; Tanjoni, I.; Carmona, A. K. [UNIFESP]; Alves, M. F. M. [UNIFESP]; Takehara, H. A.; Rucavado, A.; Ramos, O. H. P.; Moura-da-Silva, A. M.; Gutierrez, J. M.; Inst Butantan; Universidade Federal de São Paulo (UNIFESP); Univ Costa RicaBaP1 is a P-I class of Snake Venom Metalloproteinase (SVMP) relevant in the local tissue damage associated with envenomations by Bothrops asper, a medically-important species in Central America and parts of South America. Six monoclonal antibodies (MoAb) against BaP1 (MABaP1) were produced and characterized regarding their isotype, dissociation constant (K(d)), specificity and ability to neutralize BaP1-induced hemorrhagic and proteolytic activity. Two MABaP1 are IgM, three are IgG1 and one is IgG2b. the K(d)s of IgG MoAbs were in the nM range. All IgG MoAbs recognized conformational epitopes of BaP1 and B. asper venom components but failed to recognize venoms from 27 species of Viperidae, Colubridae and Elapidae families. Clone 7 cross-reacted with three P-I SVMPs tested (moojeni protease, insularinase and neuwiedase). BaP1-induced hemorrhage was totally neutralized by clones 3, 6 and 8 but not by clone 7. Inhibition of BaP1 enzymatic activity on a synthetic substrate by MABaP1 was totally achieved by clones 3 and 6, and partially by clone 8, but not by clone 7. in conclusion, these neutralizing MoAbs against BaP1 may become important tools to understand structure-function relationships of BaP1 and the role of P-I class SVMP in snakebite envenomation.(C) 2010 Elsevier B.V. All rights reserved.
- ItemSomente MetadadadosMonoclonal-antibodies specific for the free alpha-subunit of glycoprotein hormones and their use in the development of a sensitive immunofluorometric assay(Assoc Bras Divulg Cientifica, 1995-06-01) Vieira, Jose Gilberto Henriques [UNIFESP]; Nishida, Sonia Kiyomi [UNIFESP]; Lombardi, Maria Tereza; Abucham, Julio [UNIFESP]; Kasamatsu, Teresa Sayoko [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Glycoprotein hormone free alpha subunit has been used as a marker for some pituitary tumors and to study the reactivity of glycoprotein hormone-producing cells under different circumstances. We describe a highly sensitive and specific immunofluorometric assay for the measurement of serum free alpha subunit levels. The assay is based on a monoclonal antibody, specific for free alpha subunit, bound to microtiter plates. As tracer antibody we employed an europium-labelled free/complexed alpha subunit specific monoclonal antibody. Using overnight incubation and 50-mu l samples, the least detectable dose was of the order of 4 ng/l. Cross-reactivity with LH, TSH, FSH and hCG was 6.5, 1.2, 4.3 and 1.1%, respectively. Normal adult males showed values ranging from 120 to 790 ng/l, not different from normal adult premenopausal females (88 to 604 ng/l). In post-menopausal females, serum concentrations were significantly higher, ranging from 341 to 4071 ng/l. In 56 patients with untreated pituitary tumors (18 ''non-secreting'', 25 GH-producing and 13 prolactin-producing tumors), 10 showed high values, 3 of them from the first group, 3 from the second and 4- from the third. We conclude that this highly sensitive assay can be a valuable tool for the diagnosis and followup of selected patients with pituitary tumors and in other circumstances in which the glycoprotein hormone-producing cells of the pituitary require evaluation.
- ItemSomente MetadadadosRetinal and Ocular Toxicity in Ocular Application of Drugs and Chemicals - Part II: Retinal Toxicity of Current and New Drugs(Karger, 2010-01-01) Penha, Fernando Marcondes [UNIFESP]; Rodrigues, Eduardo Buchele [UNIFESP]; Maia, Mauricio [UNIFESP]; Furlani, Bruno de Alburquerque [UNIFESP]; Regatieri, Caio Vinicius Saito [UNIFESP]; Melo, Gustavo Barreto de [UNIFESP]; Magalhães, Octaviano [UNIFESP]; Manzano, Roberta [UNIFESP]; Farah, Michel Eid [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Aims: Retinal pharmacotherapy has gained great importance for the treatment of various retinal diseases. An increasing number of drugs have been constantly released into the market, especially for wet age-related macular disease and diabetic macular edema. in this review, the issues concerning the toxicity of current and new classes of drugs are discussed. Methods: An extensive search of the literature was performed to review various aspects of drug toxicity in retinal pharmacotherapy. the different major classes of drugs, such as corticosteroids, antibiotics, antimetabolites, antineoplastic agents, monoclonal antibodies (mAbs), nonsteroidal anti-inflammatory drugs, enzymes, fibrinolytics, miscellaneous anti-inflammatory and antiangiogenic agents, as well as toxicity unrelated to the drug were identified and discussed. Results: Corticosteroids like fluocinolone, dexamethasone or triamcinolone at low dose cause little damage to the retina, but at high doses signs of toxicity have been well documented. Complications like cataract and glaucoma are quite common with corticosteroids. Aminoglycosides showed differences in the type and doses associated with toxic reactions, thereby the following order of toxicity can be described (from most toxic to least toxic): gentamicin > netilmicin = tobramycin > amikacin = kanamycin. Vancomycin at the usual dose of 1 mg is not toxic to the retina, while further studies are necessary in order to clarify the safety of new-generation quinolones. 5-Fluorouracil has been shown to be nontoxic to the retina after an injection of 2.5 mg in animals. mAbs like ranibizumab and bevacizumab were demonstrated to be safe to the retina in cell culture, animals and humans at high doses. the exact biocompatibility of nonsteroidal anti-inflammatory agents like diclofenac needs further evaluation. Preservatives like benzyl alcohol and changes in pH or osmolarity exert an influence on the toxic effects of intravitreally applied drugs. Conclusions: A great number of drugs are now used mainly intravitreally without relevant retinal toxicity. Copyright (C) 2010 S. Karger AG, Basel
- ItemAcesso aberto (Open Access)Schistosoma mansoni: Description of a Potentially Useful Monoclonal Antibody that Recognizes Soluble Egg Antigen (SEA)(Instituto de Medicina Tropical, 1997-11-01) Nakhle, Maria Cristina; Carneiro, Celia Regina Whitaker [UNIFESP]; Instituto de Medicina Tropical de São Paulo; Universidade Federal de São Paulo (UNIFESP)
- ItemSomente MetadadadosA specific and highly sensitive time-resolved fluoroimmunoassay for human proinsulin(Assoc Bras Divulg Cientifica, 1996-02-01) Dalbosco, Ivaldir Sabino [UNIFESP]; Vieira, Jose Gilberto Henriques [UNIFESP]; Nishida, Sonia Kiyomi [UNIFESP]; Lombardi, Maria Tereza; Moises, Regina Celia Mello Santiago [UNIFESP]; Coifman, Renee [UNIFESP]; Russo, Ewaldo Mario Kuhlmann [UNIFESP]; SECOES ENDOCRINOL & DESENVOLVIMENTO TECN; Universidade Federal de São Paulo (UNIFESP)We describe a time-resolved fluoroimmunoaaaay specific for human Key words proinsulin using a combination of two high-affinity monoclonal antibodies, one against insulin and the other specific for intact proinsulin and for split 65-66 and des 64-65 proinsulin forms. The assay employs only 200 mu l of serum, with a detection limit of 0.1 pmol/l. The intra-assay variation coefficient was less than 3% between 3 and 1000 pmol/l, There was 0% cross-reaction with insulin, C-peptide, split 32-33 and des 31-32 proinsulin. Serum concentration of proinsulin was analyzed in 50 subjects during an oral glucose tolerance test (10 non-obese controls, 10 obese controls, 10 subjects with impaired glucose tolerance, 10 patients with type II diabetes mellitus (DM) and fasting blood glucose (FBG) <140 mg/dl, and 10 patients with type II DM and FBG >150 mg/dl), Mean fasting serum proinsulin levels measured by this assay in non-obese controls (0.84 +/- 0.90 pmol/l; 0.1-2.4 pmol/l) were lower than the results reported by other investigators. There was an increase of proinsulin related to obesity and increased glucose levels, suggesting that proinsulin levels increase with insulin resistance.
- ItemSomente MetadadadosTherapeutic monoclonal antibodies in ophthalmology(Elsevier B.V., 2009-03-01) Rodrigues, Eduardo B. [UNIFESP]; Farah, Michel E. [UNIFESP]; Maia, Mauricio [UNIFESP]; Penha, Fernando M. [UNIFESP]; Regatieri, Caio [UNIFESP]; Melo, Gustavo B. [UNIFESP]; Pinheiro, Marcelo M. [UNIFESP]; Zanetti, Carlos R.; Universidade Federal de São Paulo (UNIFESP); Universidade Federal de Santa Catarina (UFSC)Monoclonal antibodies (mAbs) can be used therapeutically by binding to molecular targets with high specificity. Therefore, they have excellent therapeutic applications in ophthalmology. This manuscript presents four aspects of the therapeutic use of mAbs in ophthalmology: the scientific rationale, the unique characteristics of selected mAbs, the current state-of-the-art application, and relevant therapeutic mAbs for future applications in ophthalmology. We identified in the literature various single-agent therapies that inhibit the following targets: tumor necrosis factor (TNF), epithelial growth factor receptor, vascular endothelial growth factor (VEGF) receptor, basic fibroblast growth factor receptor, platelet-derived growth factor, and cluster of differentiation antigens. the roles of all biochemical targets in ocular diseases were evaluated. Current and future mAbs against various cytokines were assessed for the treatment of ocular diseases. the medical literature showed the clinical benefits of mAbs for treating angiogenic and inflammatory ocular diseases. Two anti-VEGF mAbs, bevacizumab and ranibizumab, and three anti-TNF agents, infliximab, etanercept, and adalimumab, control Ocular neovascularization and intraocular inflammation. Other mAbs such as rituximab, daclizumab, efalizumab, and alemtuzumab showed positive results in animal and early clinical studies and may represent useful adjuvant therapies for ocular lymphoma or ocular inflammation. Ranibizumab is the only FDA-approved therapy; for other mAbs the so-called off-label application remains the standard. Intravenous administration of mAbs has demonstrated acceptable toxicity profiles, while intraocular injection may decrease the chances of systemic complications and increase the amount of drug available to the retina and choroid. in conclusion, effective clinical use of mAbs in ophthalmology is more commonly seen in the field of angiogenic vitreoretinal and autoimmune inflammatory diseases. the challenge for the future is combining biologic therapies to improve the quality and duration of responses while diminishing side effects. the role of mAbs within ophthalmic treatments will be defined according to future clinical experience and the results of randomized clinical trials. (C) 2008 Elsevier B.V. All rights reserved.