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Memórias do Instituto Oswaldo Cruz

Print version ISSN 0074-0276On-line version ISSN 1678-8060

Mem. Inst. Oswaldo Cruz vol.84 no.2 Rio de Janeiro Apr./June. 1989

http://dx.doi.org/10.1590/S0074-02761989000200003 

Evaluation of antibody responses in american visceral leishmaniasis by ELISA and immunoblot

Thomas G. Evans1 

Edward C. Krug1 

Mary E. Wilson2 

Antonio Wilson Vasconcelos3 

Joaquim Eduardo de Alencar3 

Richard D. Pearson1 

University of Virginia, School of Medicine, Departments of Medicine and Pathology. Divisions of Geographic Medicine and Infectious Diseases, Charlottesville, USA

University of Iwoa, School of Medicine, Department of Internal Medicine. Division of Infectious Diseases, Iwoa, USA

Universidade Federal do Cear¨¢, N¨²cleo de Medicina Tropical, Fortaleza, Brasil

ABSTRACT

American visceral leishmaniasis (AVL) is an important disease among children of northeast Brazil. In order to characterize antibody responses during AVL, sera of hospitalized patients were analyzed by ELISA and Western blot using a Leishmania chagasi antigen preparation. The ELISA was positive (asorbance [greater than or equals to] 0.196) at a serum dilution of 1:1024 in all patients at presentation, and fell to ward control levels over the following year. Only one of 72 control subjects tested positive, and that donor had a sibling with AVL. Immunoblots of the patients' sera recognized multiple bands, the most frequent of which were at approximately 116 kDa, 70 kDa, and 26 kDa. Less frenquently observed were bands at approximately 93 kDa, 74 kDa, 62 kDa, 46 kDa and 32 kDa. The ELISA responses and patterns of banding were distinctive for AVL, and could be used to differentiate patients with AVL from those with Chagas' disease of cutaneous leishmaniasis. Sera from six AVL patients followed for up to six weeks after treatment identified no new bands. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of surface iodinated parasite proteins showed one major band and four minor bands, whereas SDS-PAGE of biotinylated prarasite proteins revealed a banding pattern similar to those of patient sera. AVL appears to produce characteristic immunoblot patterns which can be used along with a sensitive screening ELISA to diagnose AVL.

Key words: Visceral leishmaniasis; Leishmania donovani chagasi; ELISA; immunoblot

RESUMO

A leishmaniose visceral americana é uma doença importante entre crianças do nordeste do Brasil. Com o objetivo de caracterizar a resposta imune humoral durante o curso da doença, foram analisados soros de pacientes hospitalizados, pelos métodos ELISA e Western blot, usando-se preparaçöes de antígenos de Leishmania donovani chagasi. O teste de ELISA foi positivo (absorbância > 0,196), numa diluiçäo de 1: 1024, em todos os pacientes no início da doença, diminuindo no decorrer do ano, para os níveis dos indivíduos controles. Apenas um dos 72 controles, que tinha um irmäo com leishmaniose visceral, apresentou positividade para o teste. "Immunoblots" de soros de pacientes apresentaram bandas múltiplas, sendo as mais freqüentes de aproximadamente 116 kDa, 70 kDa. Com menor freqüência foram observadas bandas de 93 kDa, 74 kDa, 62 kDa, 46 kDa e 32 kDa. As respostas, através de ELISA e dos padröes de separaçäo por "immunoblot", foram distintas na leishmaniose visceral e podem ser usadas para diferenciar pacientes com leishmaniose visceral daqueles com doenças de Chagas ou leishmaniose cutânea. As amostras sorológicas de pacientes acompanhados durante seis semanas após o tratamento näo apresentaram nenhuma banda nova. A eletroforese em gel de poliacrilamida-sulfato de dodecil de sódio (SDS-PAGE) das proteínas iodinadas da superfície do parasita mostrou uma banda principal e quatro menores, enquanto a eletroforese de proteínas biotiniladas do parasita mostrou um padräo semelhante aquele apresentado pelos soros dos pacientes. A leishmaniose visceral parece produzir um padräo de "immunoblot" característico que pode ser usado, juntamente com um método sensível tipo ELISA, no diagnóstico de leishmaniose visceral americana.

Palavras-Chave: leishmaniose visceral; Leishmania donovani chagasi; ELISA; "Immunoblot"

 

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