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A new tool of enterotoxigenic Escherichia coli diagnosis: recombinant antibodies against heat-stable toxin.

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Author(s):
Caio Raony Farina Silveira
Total Authors: 1
Document type: Master's Dissertation
Press: São Paulo.
Institution: Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI)
Defense date:
Examining board members:
Roxane Maria Fontes Piazza; Geraldo Santana Magalhães; Rosa Maria Silva
Advisor: Roxane Maria Fontes Piazza
Abstract

Recombinant antibodies have been used as diagnostic tools since they can be produced at low cost and on a large scale. A synthetic gene encoding an antibody fragment (scFv) specific for the heat-stable toxin (ST) with optimized codon for Escherichia coli expression was employed. This gene was amplified in the cloning vector and subcloned into pET28a expression vector. E. coli BL21(DE3) cells were transformed with the recombinant plasmid and induced. Large amounts of antibody fragment were found in the insoluble fraction. Thus it is submitted to nickel-affinity chromatography in urea presence, followed by refolding step. By immunofluorescence assay and ELISA, the obtained antibody showed to be functional with no cross-reaction to the negative controls. Furthermore, it was stable when stored at 4 °C, therefore a promising tool for ETEC diagnosis detecting the ST toxin. (AU)

FAPESP's process: 10/14659-6 - Obtention and characterization of recombinant antibodies (scFv) against heat-stable (st) toxin produced by Enterotoxigenic Escherichia coli (ETEC)
Grantee:Caio Raony Farina Silveira
Support Opportunities: Scholarships in Brazil - Master