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Desenvolvimento de anticorpos recombinantes anti-Schistosoma.
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Orientador(es)
Resumo(s)
A schistosomose é uma parasitose causada por tremátodes parasitas do género
Schistosoma. Trata-se de uma doença tropical negligenciada, em que quase 240 milhões
de pessoas estão infetadas em todo o mundo e cerca de 700 milhões estão em risco de
contrair a infeção. A transmissão já foi relatada em 78 países.
O diagnóstico é fundamental para a erradicação da doença. No entanto, as técnicas
de diagnóstico podem apresentar algumas limitações de sensibilidade, principalmente em
infeções leves, quando há baixa excreção de ovos.
Assim, o presente estudo teve como objetivo desenvolver anticorpos
recombinantes contra antigénios de Schistosoma mansoni.
Parasitas adultos de S. mansoni foram extraídos através da perfusão do fígado de
murganhos e mantidos em stock num tampão para conservação para serem utilizados nas
diversas técnicas deste trabalho. Selecionaram-se anticorpos pela técnica de Phage
Display, com 3 ciclos de seleção por Biopanning; a visualização da ligação antigénio anticorpo foi feita através do teste de ELISA Indireta; a visualização da reação ao
substrato foi feita através do teste de Imunohistoquímica; a amplificação do ADN foi feita
por técnica de PCR e também foi realizada a Sequenciação Genética.
Os dois primeiros ciclos de seleção do Biopanning apresentaram valores positivos
para a presença de fagos, mas o terceiro ciclo não gerou quantidade significativa de
partículas fágicas. Quanto ao teste de ELISA indireta, observou-se que a maioria dos
anticorpos reagiu igualmente para os antigénios de macho e fêmea. Nas reações do teste
de Imunohistoquímica, não houve diferenças significativas entre os grupos teste e
controlo. Foram selecionadas 10 colónias para amplificação do ADN por PCR, e destas,
3 amostras que apresentaram bandas entre 700 e 900 pares de base foram enviadas para
sequenciação genética. Apenas numa das amostras foi possível identificar algumas
regiões das cadeias pesada e leve e o linker, portanto, seriam necessários alguns ajustes
nos protocolos adotados para obtenção de um resultado mais satisfatório.
Schistosomiasis is a parasitic disease caused by parasitic trematodes of the genus Schistosoma. It is a neglected tropical disease, in which almost 240 million people are infected worldwide and about 700 million are at risk of contracting the infection. Transmission has been reported in 78 countries. An accurate diagnosis is essential for the eradication of the disease. However, diagnostic techniques may have some sensitivity limitations, especially in mild infections, with low egg rates. Therefore, the present study aimed to develop recombinant antibodies against Schistosoma mansoni antigens. Adult parasites of S. mansoni were extracted by perfusion of the liver of mice and kept in conservation buffer to be used in different techniques during this study. The methodology was based on Phage Display Technology, with 3 rounds of selection by Biopanning; Visualization of the antigen-antibody binding was performed through Indirect ELISA test; Visualization of the substrate reaction was performed through the Immunohistochemistry test, Amplification of DNA was performed by PCR technique, and it was performed Genetic Sequencing. The first two selection rounds of Biopanning showed positive values for the presence of phages, but the third cycle did not generate a significant amount of phage particles. For the indirect ELISA test, it was observed that most of the antibodies reacted equally to the male and female antigens. In the reactions of the Immunohistochemistry test, there were no significant differences between the test and control groups. 10 colonies were selected for DNA amplification by PCR, and of these, 3 samples that showed bands between 700 and 900 base pairs were sent for genetic sequencing. Only in one of the samples it was possible to identify some regions of the heavy and light chains and the linker, therefore, some adjustments in the adopted protocols would be necessary to obtain a more satisfactory outcome.
Schistosomiasis is a parasitic disease caused by parasitic trematodes of the genus Schistosoma. It is a neglected tropical disease, in which almost 240 million people are infected worldwide and about 700 million are at risk of contracting the infection. Transmission has been reported in 78 countries. An accurate diagnosis is essential for the eradication of the disease. However, diagnostic techniques may have some sensitivity limitations, especially in mild infections, with low egg rates. Therefore, the present study aimed to develop recombinant antibodies against Schistosoma mansoni antigens. Adult parasites of S. mansoni were extracted by perfusion of the liver of mice and kept in conservation buffer to be used in different techniques during this study. The methodology was based on Phage Display Technology, with 3 rounds of selection by Biopanning; Visualization of the antigen-antibody binding was performed through Indirect ELISA test; Visualization of the substrate reaction was performed through the Immunohistochemistry test, Amplification of DNA was performed by PCR technique, and it was performed Genetic Sequencing. The first two selection rounds of Biopanning showed positive values for the presence of phages, but the third cycle did not generate a significant amount of phage particles. For the indirect ELISA test, it was observed that most of the antibodies reacted equally to the male and female antigens. In the reactions of the Immunohistochemistry test, there were no significant differences between the test and control groups. 10 colonies were selected for DNA amplification by PCR, and of these, 3 samples that showed bands between 700 and 900 base pairs were sent for genetic sequencing. Only in one of the samples it was possible to identify some regions of the heavy and light chains and the linker, therefore, some adjustments in the adopted protocols would be necessary to obtain a more satisfactory outcome.
Descrição
Palavras-chave
Parasitologia médica Schistosomose Schistosoma mansoni Anticorpos