RESUMO
A simple and reliable indirect enzyme-linked immunosorbent assay for detection of antibodies directed against a major bovine viral diarrhea virus (BVDV) immunogen, the E2 glycoprotein (tE2-ELISA), has been developed using the recombinant C-terminal truncated E2 glycoprotein (tE2) expressed in a Drosophila melanogaster system. This strategy demonstrated that tE2 is secreted efficiently in the supernatant, no purification steps are necessary, it is easy to produce and carries out the post translational modifications necessary to preserve its native conformation. Preliminary analysis of 183 cattle serum samples using tE2-ELISA showed a 98% specificity and a 100% sensitivity compared with the standard homologous BVDV virus neutralization test. The results also showed that the tE2 is immunoreactive because the conformation and antigenicity of the original E2 are maintained to a large extent. To our knowledge this is the first study report of the recombinant tE2 of BVDV expressed in D. melanogaster system as an antigen for ELISA.
Assuntos
Anticorpos Antivirais/sangue , Antígenos Virais/imunologia , Vírus da Diarreia Viral Bovina/imunologia , Ensaio de Imunoadsorção Enzimática , Proteínas do Envelope Viral/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Monoclonais/imunologia , Antígenos Virais/biossíntese , Antígenos Virais/isolamento & purificação , Bovinos , Vírus da Diarreia Viral Bovina/genética , Drosophila melanogaster , Testes de Neutralização , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Sensibilidade e Especificidade , Proteínas do Envelope Viral/biossíntese , Proteínas do Envelope Viral/isolamento & purificaçãoRESUMO
Experiments were performed to determine whether sialic acids are expressed in two dermatophytes: Trichophyton rubrum and T. mentagrophytes, similarly to other fungal pathogens. Chemical extraction of mycelia and microconidia followed by high-performance thin-layer chromatography and colorimetric assays were all negative for sialic acid. Incubation of dermatophytes in the presence of Limax flavus agglutinin, specific for sialic acid, was negative in a fluorescence staining test. We have also used other lectins that bind to sialic acid and/or other sugar residues, and these ligands coupled to fluorescein strongly stained these fungi. Such fluorescence staining was not abolished or reduced when fungi were pretreated with sialidase. Different strains of influenza virus which recognize sialic acid residues were also used, but no agglutination of the dermatophytes was observed. Based on these methods, which successfully revealed the presence of sialic acids in other fungal pathogens, we show that these monosaccharides do not occur in both dermatophyte species. Thus, sialic acids do not seem to play a role in the pathogenicity of these fungi.
Assuntos
Ácidos Siálicos/biossíntese , Trichophyton/metabolismo , Aglutinação , Cromatografia Líquida de Alta Pressão , Colorimetria , Técnica Direta de Fluorescência para Anticorpo , Lectinas/metabolismo , Mitocôndrias/metabolismo , Micélio/química , Micélio/metabolismo , Neuraminidase/farmacologia , Orthomyxoviridae/metabolismo , Ácidos Siálicos/análise , Especificidade da Espécie , Trichophyton/efeitos dos fármacosRESUMO
The increasing number of reports on the presence of sialic acids in fungi (N-acetyl-, N-glycolyl- and 5,9-N,O-diacetylneuraminic acids) based on direct and indirect evidence warrants the present review. Formerly suggested as sialidase-sensitive sources of anionic groups at the cell surface of fungal species grown in chemically defined media (e.g., Fonsecaea pedrosoi), sialic acids have also been found in Sporothrix schenckii, Paracoccidioides brasiliensis, Cryptococcus neoformans and recently, in Candida albicans. Methods used involved adequate hydrolysis and extraction procedures, HPTLC, gas-chromatography, colorimetry, mass spectroscopy, lectin and influenza virus binding. Apart from protecting fungal cells against phagocytosis (S. schenckii, C. neoformans) and playing a cellular structural role (F. pedrosoi), other biological functions of sialic acids are still being investigated.