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Master's Dissertation
DOI
10.11606/D.9.2003.tde-06032015-145009
Document
Author
Full name
Angélica Maria Casimiro
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2003
Supervisor
Committee
Kanamura, Herminia Yohko (President)
Pinto, Pedro Luiz Silva
Vaz, Adelaide Jose
Title in Portuguese
Padronização e avaliação de método sorológico ELISA para detecção de anticorpos IgG anti-Cryptosporidium sp
Keywords in Portuguese
Cryptosporidium
ELISA (Avaliação)
Parasitologia
Testes imunológicos (Testes)
Abstract in Portuguese
O presente trabalho teve como objetivo padronizar a técnica de ELISA para detecção de anticorpos IgG anti-Cryptosporidium sp para aplicação em estudos epidemiológicos da criptosporidiose em imunocompetentes. Para obtenção de antígeno, bezerros foram oralmente infectados. Os oocistos foram recuperados das fezes doanimal, com a utilização do gradiente de sacarose modificado, técnica de concentração onde se obteve o melhor rendimento. Para preparação do antígeno, os oocistos foram rompidos através de ciclos de congelamento/descongelamento e ultra-som. Soros controle positivo foram escolhidos entre o grupo de funcionários do laboratório de Parasitologia, pois apresentavam anticorpos anti-Cryptosporidium e devido as suas atividades no laboratório era um grupo mais exposto; soros controle negativo foram escolhidos entre aqueles com leituras de densidade óptica menores que 0,300 no ELISA para detecção de anticorpos anti-Cryptosporidium. Diferentes grupos de soros de indivíduos clinicamente normais (funcionários da parasitologia, doadores de sangue, pacientes que fizeram o Pré-Natal) ou outras infecções parasitárias (cisticercose, toxoplasmose, esquistossomose, Doença de Chagas, leishmaniose), foram avaliados para presença de anticorpos anti-Cryptosporidium. A alta freqüência foi observada para o grupo de pacientes com Doença de Chagas (66,6%) e baixa freqüência para o grupo de pacientes com esquistossomose e toxoplasmose (20,0%). A especificidade do teste ELISA para Cryptosporidium foi demonstrada com significante redução nas leituras de 0.0. observada em alguns soros após absorção dos anticorpos anti-Cryptosporidium. Além disso, no grupo de pacientes Pré-Natal 14,6%, quando comparada a alta freqüência de anticorpos anti-Cryptosporidium 52,0%, indica provável ausência de reações cruzadas entre os dois antígenos. Enfim, os resultados obtidos sugerem que a técnica de ELISA pode ser uma importante metodologia para aplicação em estudos soroepidemiológicos da criptosporidiose.
Title in English
Standardization and evaluation of serological method ELISA for detection of IgG anti-Cryptosporidium sp
Keywords in English
Cryptosporidium
ELISA (Review)
Immunological tests (Tests)
Parasitology
Abstract in English
The aim of the present study was to standardize an immunoenzymatic assay, ELISA, for detection of IgG antibodies to Cryptosporidium sp for use in epidemiologic studies on cryptosporidiosis. For antigen preparation, oocysts were obtained from fecal samples of orally infected calves. A modified sucrose gradient, concentration technique was used for recovered and purification of oocysts, which were ruptured by using freezethaw cycles and ultra-sonication. Positive control sera were chosen among the Parasitology workers, who presented anti-Cryptosporidium antibodies and had been exposed to this parasite, because of their activities in the laboratory; and negative control sera were chosen among the ones with optical density (O.D.) readings lower than 0,300 at ELISA for anti- Cryptosporidium antibodies. Oifferent groups of sera from clinically normal individuais (parasitology workers, blood donors, pregnant patients) or with other parasite infection (cysticercisis, toxoplasmosis, schistosomiasis, Chagas disease, leishmaniasis) were evaluated for the presence of Cryptosporidium antibodies. The higher frequency was observed for the group of patients with Chagas disease (66.6%) and the lower frequency for the group patients with schistosomiasis and toxoplasmosis (20.0%). The specificity of the Cryptosporidium-ELISA test was demonstrated when significant reduction of the 0.0. readings was observed for some serum samples after absorption of the anti-Cryptosporidium antibodies. Also, in the group of pregnant patients, the high frequency of 52.0% for anti-Cryptosporidium antibodies when compared to the low frequency of 14.6% for anti-Toxoplasma antibodies might suggest possible absence of cross reactions between these two closely related parasite antigens. The ELISA for detection of anti-Cryptosporidium antibodies, as standardized in the present work, can constitute a good toel for epidemiological studies of cryptosporidiosis.
 
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Publishing Date
2015-03-06
 
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