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Doctoral Thesis
DOI
https://doi.org/10.11606/T.10.2018.tde-12032018-111930
Document
Author
Full name
Tatiana Barrionuevo Gotti
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2015
Supervisor
Committee
Aniz, Patricia Antonia Estima Abreu de (President)
Brandão, Paulo Eduardo
Moreno, Andrea Micke
Piazza, Roxane Maria Fontes
Rocha, Letícia Barboza
Title in Portuguese
Desenvolvimento de um teste dipstick para o diagnóstico da leptospirose animal
Keywords in Portuguese
Diagnóstico
Leptospira
LipL32
Proteínas recombinantes
Teste imunocromatográfico
Abstract in Portuguese
A leptospirose é uma doença bacteriana infectocontagiosa, de curso agudo ou crônico, causada por espiroquetas do gênero Leptospira, de caráter zoonótico e cosmopolita que acomete o homem e os animais domésticos e silvestres. Pode ser transmitida de forma direta pelo contato com os fluidos contendo leptospiras, através das vias transplacentária e hematogênica, genital e o ato de mamar; ou de forma indireta pelo contato com ambiente contaminado com leptospiras. O conhecimento da gravidade da infecção, da distribuição geográfica, dos fatores de risco e das estirpes circulantes é de extrema importância para o estabelecimento da epidemiologia e o aprimoramento de medidas preventivas e diagnósticas. Neste estudo, avaliou-se a utilização de proteínas recombinantes de Leptospira spp. como antígenos no desenvolvimento de um teste rápido baseado em ensaio imunocromatográfico do tipo dipstick, como método diagnóstico da leptospirose animal. Foram selecionadas 11 proteínas recombinantes como candidatos a antígenos. As proteínas recombinantes purificadas foram avaliadas na detecção de anticorpos específicos por Western-blotting e ELISA. Somente a LipL32 apresentou reatividade com os soros positivos para leptospirose. Dois testes imunocromatográficos, utilizando a proteína LipL32, foram desenvolvidos. Um teste tipo I utilizando a proteína LipL32 conjugada ao ouro coloidal e outro tipo III com o ouro coloidal conjugado a proteína A e a LipL32 na linha teste. Em ambos as linhas teste e controle reagiram, demonstrando que os testes estão funcionando. O teste tipo I realizado manualmente mostrou resultados satisfatórios para os soros bovinos e soro hiperimune de coelho. O tipo III mostrou reatividade para as amostras de soro bovino, equino, coelho e cão. Quando se aplicou este dois testes em máquinas automatizadas não foi possível detectar a linha teste, provavelmente devido a necessidade de nova padronização de todos os parâmetros do método.
Title in English
Development of a dipstick test for the diagnosis of animal leptospirosis
Keywords in English
Diagnosis
Immunoassay test
Leptospira
LipL32
Recombinant proteins
Abstract in English
Leptospirosis is an infectious bacterial disease of acute or chronic course, caused by spirochetes of the genus Leptospira, with zoonotic and cosmopolitan character, which affects humans, wild and domestic animals. It can be transmitted directly by contact with fluids containing leptospires through placental, hematogenous and genital routes and through the act of breastfeeding; or indirectly by contact with an environment contaminated with leptospires. Acquiring knowledge about the infection severity, the geographical distribution, the risk factors, and the circulating strains is of utmost importance to stablish the epidemiology and to improve preventive and diagnostic measures. In this study, recombinant proteins of Leptospira spp. were evaluated as antigens in the development of a rapid immunochromatographic assay based on the type dipsticks a method of diagnosing animal leptospirosis. 11 recombinant proteins were selected as candidate antigens. The purified recombinant proteins were evaluated in the detection of specific antibodies by Western blotting and ELISA. Only the LipL32 protein showed reactivity with positive sera for leptospirosis. Two immunochromatographic tests, using LipL32 protein, have been developed: a type I test using the LipL32 protein conjugated to colloidal gold and another, type III with colloidal gold conjugated to protein A and LipL32 in the test line. In both assays the test lines and the control lines reacted, showing that the methods are working. The type I test performed manually showed satisfactory results for bovine and hyperimmune rabbit sera. The type III test showed reactivity for bovine, horse, rabbit and dog sera. When these two tests were applied in automated machinery, it has not been possible to detect the line test, probably due to the need for further standardization of all method parameters.
 
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Release Date
2020-06-11
Publishing Date
2018-07-25
 
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