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Expression and purification optimization of recombinant antibody (scFv) against heat-labile toxin (LT) of enterotoxigenic Escherichia coli (ETEC)

Grant number: 11/24111-0
Support Opportunities:Scholarships in Brazil - Scientific Initiation
Effective date (Start): March 01, 2012
Effective date (End): December 31, 2012
Field of knowledge:Biological Sciences - Microbiology - Applied Microbiology
Principal Investigator:Roxane Maria Fontes Piazza
Grantee:Danielle Dias Munhoz
Host Institution: Instituto Butantan. Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil


Diarrheagenic Escherichia coli (DEC) is responsible for several cases of acute diarrhea, especially among children in developing countries, with important public health effects. Among the pathotypes of DEC, can be highlighted enterotoxigenic E. coli (ETEC) producer of heat-labile toxin (LT), and heat-stable toxin (ST), one of the most frequent pathotypes of diarrheagenic E. coli. Indeed, the diagnosis is essential in order to prevent possible outbreaks and determine the best method of treatment. Immunological tests have several advantages compared to other detection methods, such as high specificity, sensibility, easy sample preparation and to perform, besides the rapid achievement of results. Antibodies generated from the natural immune response or through immunization (polyclonal) are presented as a molecules mixture with different affinities and specificities, being produced in limited quantities. On the other hand, monoclonal antibodies compared to polyclonal antibodies, exhibit homogeneity, and high specificity and are produced indefinitely, however, require specialized cell culture and extensive involvement of time and effort. Genetic engineering has been used to obtain recombinant antibodies, in order to maintain homogeneity and specificity of monoclonal antibodies, with large-scale production, low cost, and the construction development that maintains or improves the functional properties of an antibody. Currently, it is possible to produce recombinant proteins, like antibody fragments, using different expression systems. One of the methodologies used is the cloning of variable domains of light chains (VL) and heavy chains (VH) fused to an immunoglobulin by a flexible linker, allowing the correct interaction between the domains and the preservation of the antigen-binding site called ScFv ( Single Chain Variable Fragment). Because of the limiting polyclonal antibody production and the extensive time and labor involved in the monoclonal antibody production emerged the aim of this work, is the optimizing the expression and purification of the construction of ScFv pMST3-LT for its use as a tool in the immunodiagnosis of ETEC strains.(AU)

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Scientific publications
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
OZAKI, CHRISTIANE Y.; SILVEIRA, CAIO R. F.; ANDRADE, FERNANDA B.; NEPOMUCENO, ROBERTO; SILVA, ANDERSON; MUNHOZ, DANIELLE D.; YAMAMOTO, BRUNO B.; LUZ, DANIELA; ABREU, PATRICIA A. E.; HORTON, DENISE S. P. Q.; et al. Single Chain Variable Fragments Produced in Escherichia coli against Heat-Labile and Heat-Stable Toxins from Enterotoxigenic E-coli. PLoS One, v. 10, n. 7, . (11/12928-2, 10/14659-6, 11/24111-0, 04/08297-3)

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