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Standardization of the capture elisa for quantification of heat-labile enterotoxin expressed by enterotoxigenic escherichia coli strains isolated from humans

Melissa Ang Simões Lásaro J. F Rodrigues; Luís Carlos de Souza Ferreira; Congresso do Instituto de Ciências Biomédicas (4. 2005 São Paulo)

Resumos São Paulo: Comissão de Cultura e Extensão Universitária do ICB/USP, 2005

São Paulo Comissão de Cultura e Extensão Universitária do ICB/USP 2005

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  • Título:
    Standardization of the capture elisa for quantification of heat-labile enterotoxin expressed by enterotoxigenic escherichia coli strains isolated from humans
  • Autor: Melissa Ang Simões Lásaro
  • J. F Rodrigues; Luís Carlos de Souza Ferreira; Congresso do Instituto de Ciências Biomédicas (4. 2005 São Paulo)
  • Assuntos: MICROBIOLOGIA
  • É parte de: Resumos São Paulo: Comissão de Cultura e Extensão Universitária do ICB/USP, 2005
  • Notas Locais: Disponível somente em CD-ROM
  • Descrição: Enterotoxigenic Escherichia coli (ETEC) represent one of the most important etiological agents of diarrhea in developing countries. The pathogenesis of ETEC is closely related to the ability to colonize the gut epithelium and produce at least one of two enterotoxins: the heat-labile toxin (LT) and the heat stable toxin (ST). Detection of LT-producing ETEC strains have been performed by different immunological approaches including the Biken test and capture assays such as the GM1 enzyme linked immunosorbent assay (GM1-ELISA) and the antibody capture ELISA (cELISA). Both GM1- and cELISA have been also used to quantify the amount of toxin produced by toxigenic strains either in culture supernatants or whole-cell extracts. However, unlike the GM1-ELISA, the cELISA has not been previously standardized for maximal sensitivity both for quantitative and qualitative purposes. In this work we evaluated different parameters affecting sensitivity of the sandwich assay for LT quantification both purified LT and whole cell extracts of the ETEC H10407 strain. The concentrations of LT-specific antibodies used in the capture and detection steps proved to key steps for the sensitivity of the assay. Dilution curves of the capture (anti-cholera toxin serum raised in rabbit) and detection (anti-LT serum raised in mice) antibodies showed maximal sensitivity at dilutions of 1/1,000 and 1/5,000 for the capture (anti-CT titer of 3.3 x 105) and detection (anti-LT titer of 1 x 105) serum,
    respectively. Under optimal experimental conditions, a maximal sensitivity of 2 ng/mL of purified toxin as achieved with the cELISA, while the GM1-ELISA could detect LT concentrations up to 0.5 ng/ml. The standardized procedure proved to be a reliable method for the quantification of LT in culture supernatants and whole-cell extracts of ETEC. The present results indicate that the cELISA represents a rather simple and sensitive method both for detection and quantification of LT produced by ETEC strains
  • Editor: São Paulo Comissão de Cultura e Extensão Universitária do ICB/USP
  • Data de criação/publicação: 2005
  • Formato: res. 49.
  • Idioma: Inglês
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  • Realização: ABCD-USP USP   Logos de Redes Sociais: Freepik

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