Dificuldades na obtenção e caracterização de anticorpos monoclonais murinos anti-proteína F recombinante do vírus (RSV) para diagnóstico laboratorial
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Data
2009-02-27
Autores
Souza, Aparecida Vitória Gonçalves de [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
O Vírus Sincicial Respiratório humano (VSR) é o principal agente causal das Infecções Respiratórias Agudas (IRAs) em lactantes e pré-escolares. Apresenta dois subgrupos – A e B – sendo o primeiro responsável por quadros clínicos mais graves. Os RNA mensageiros virais codificam 11 proteínas conhecidas, das quais a proteína F é responsável pela fusão viral à célula e se apresenta na forma de F0 com peso molecular de 67kDa, podendo ser clivada em duas unidades: F2 de 20kDa e a F1 de 47kDa. A proteína F recombinante (Fr), expressa em E.coli (BL21A no vetor pET28a), foi cedida por pesquisadores da UNESP de São José do Rio Preto. Com intuito de produzir anticorpos monoclonais contra a proteína Fr para fins de diagnóstico laboratorial, inoculamo-la em camundongos isogênicos Balb/c. Durante o desenvolvimento do monoclonal, foi necessário a eliminação da contaminação de cultura por Mycoplasma ssp. Após ajuste de dose de inoculação e descontaminação do antígeno (hipótese: contaminação com endotoxinas provenientes de E.coli), seis clones secretores de anticorpos monoclonais foram produzidos (5 do tipo IgM e 1 do tipo IgG2a) e testados, simultâneamente, contra toxina bacteriana e proteína Fr (40μg/mL) por técnica de ELISA indireto. Paralelamente, testes por citometria de fluxo foram realizados, incubando-se os clones obtidos com E.coli (bactéria com membrana permeabilizada e in natura). Os clones (VIRSV2-87A74 e VIRSV2-87A80) apresentam um reconhecimento inespecífico de proteínas da E. coli e não foi possível caracterizar os clones obtidos em função da dificuldade de se obter novas amostras de proteína Fr. Para o futuro, os anticorpos obtidos deverão ser marcados com isotiocinato de fluoroceína e comporem um amplo teste epidemiológico em paralelo com o kit disponível em mercado respeitando a sazonalidade da doença. Novos ensaios para caracterização...
The Human Respiratory Syncytial Virus (HRSV) is the main cause of Acute Respiratory Insufficiency in suckling child and children below 5 years old. It has 2 subgroups – A and B – being the first one responsible for more dangerous clinical situations. The viral messenger RNA codifies 11 known proteins, which the F protein is responsible for the viral fusion and it is presented as F0 with molecular weight of 67kDa, but once is broke it generates two units: F2 with 20kDa and F1 with 47kDa. The recombinant F (Fr) protein were expressed on E.coli (BL21A, on vector pET28a), and it was given to us by researches from UNESP of São José do Rio Preto. Wishing to produce monoclonal antibodies (Mab) against Fr protein to laboratorial diagnosis, mice (Balb/c) were inoculated with de antigen (Fr protein). During the development of the Mab, it was necessary to eliminate, from the culture, Mycoplasma spp. After we adjusted the necessary amount to inoculate on the mice and eradicated the contamination of the antigen (hypotheses: there were endotoxins from the E.coli), six clones that secrete Mab were produced (5 of the kind IgM, and 1 of the kind IgG2a) and they were tested, simultaneously, against bacterium toxin and Fr protein (40μg/mL) using ELISA technique. Together, flow cytometry test were performed when we incubated the clones with E. coli (bacterium with permeable membrane and in natura). The clones (VIRSV2-87A74 and VIRSV2-87A80) presented an unspecific recognition for the proteins from E.coli and it was not possible to characterize the cells because we couldn´t get more samples of Fr protein. For the future, the Mab should be conjugated with FITC and then, they must be part of a wide epidemiologic test side by side from the commercial kit, always respecting the seasonal disease. New assays to characterize antibodies (like Western blotting and imunepreciptation) are already being done... (Complete abstract click electronic access below)
The Human Respiratory Syncytial Virus (HRSV) is the main cause of Acute Respiratory Insufficiency in suckling child and children below 5 years old. It has 2 subgroups – A and B – being the first one responsible for more dangerous clinical situations. The viral messenger RNA codifies 11 known proteins, which the F protein is responsible for the viral fusion and it is presented as F0 with molecular weight of 67kDa, but once is broke it generates two units: F2 with 20kDa and F1 with 47kDa. The recombinant F (Fr) protein were expressed on E.coli (BL21A, on vector pET28a), and it was given to us by researches from UNESP of São José do Rio Preto. Wishing to produce monoclonal antibodies (Mab) against Fr protein to laboratorial diagnosis, mice (Balb/c) were inoculated with de antigen (Fr protein). During the development of the Mab, it was necessary to eliminate, from the culture, Mycoplasma spp. After we adjusted the necessary amount to inoculate on the mice and eradicated the contamination of the antigen (hypotheses: there were endotoxins from the E.coli), six clones that secrete Mab were produced (5 of the kind IgM, and 1 of the kind IgG2a) and they were tested, simultaneously, against bacterium toxin and Fr protein (40μg/mL) using ELISA technique. Together, flow cytometry test were performed when we incubated the clones with E. coli (bacterium with permeable membrane and in natura). The clones (VIRSV2-87A74 and VIRSV2-87A80) presented an unspecific recognition for the proteins from E.coli and it was not possible to characterize the cells because we couldn´t get more samples of Fr protein. For the future, the Mab should be conjugated with FITC and then, they must be part of a wide epidemiologic test side by side from the commercial kit, always respecting the seasonal disease. New assays to characterize antibodies (like Western blotting and imunepreciptation) are already being done... (Complete abstract click electronic access below)
Descrição
Palavras-chave
Aparelho respiratorio - Doenças - Aspectos imunológicos, Vírus respiratórios, Virus sincicial repiratório - Diagnóstico, Respiratory Syncytial Virus, Laboratorial diagnosis, Monoclonal antibodies
Como citar
SOUZA, Aparecida Vitória Gonçalves de. Dificuldades na obtenção e caracterização de anticorpos monoclonais murinos anti-proteína F recombinante do vírus (RSV) para diagnóstico laboratorial. 2009. 91 f. Dissertação (mestrado) - Universidade Estadual Paulista, Faculdade de Medicina de Botucatu, 2009.