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Single Chain Variable Fragments Produced in Escherichia coli against Heat-Labile and Heat-Stable Toxins from Enterotoxigenic E. coli.

Ozaki CY, Silveira CR, Andrade FB, Nepomuceno R, Silva A, Munhoz DD, Yamamoto BB, Luz D, Abreu PA, Horton DS, Elias WP, Ramos OH, Piazza RM - PLoS ONE (2015)

Bottom Line: These strains can produce two enterotoxins associated with the manifestation of clinical symptoms that can be used to detect these pathogens.Both antibody fragments were able to recognize the cell-interacting toxins by immunofluorescence, the purified toxins by ELISA and also LT-, ST- and LT/ST-producing ETEC strains.The developed recombinant scFvs against LT and ST constitute promising starting point for simple and cost-effective ETEC diagnosis.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Bacteriologia, Instituto Butantan, São Paulo, SP, Brasil.

ABSTRACT

Background: Diarrhea is a prevalent pathological condition frequently associated to the colonization of the small intestine by enterotoxigenic Escherichia coli (ETEC) strains, known to be endemic in developing countries. These strains can produce two enterotoxins associated with the manifestation of clinical symptoms that can be used to detect these pathogens. Although several detection tests have been developed, minimally equipped laboratories are still in need of simple and cost-effective methods. With the aim to contribute to the development of such diagnostic approaches, we describe here two mouse hybridoma-derived single chain fragment variable (scFv) that were produced in E. coli against enterotoxins of ETEC strains.

Methods and findings: Recombinant scFv were developed against ETEC heat-labile toxin (LT) and heat-stable toxin (ST), from previously isolated hybridoma clones. This work reports their design, construction, molecular and functional characterization against LT and ST toxins. Both antibody fragments were able to recognize the cell-interacting toxins by immunofluorescence, the purified toxins by ELISA and also LT-, ST- and LT/ST-producing ETEC strains.

Conclusion: The developed recombinant scFvs against LT and ST constitute promising starting point for simple and cost-effective ETEC diagnosis.

No MeSH data available.


Related in: MedlinePlus

Construction, expression and purification of scFv-LT.(A) scFv-LT amino acids sequence: VH—Heavy chain variable domain, L—Linker and VL—Light chain variable domain sequence. CDRs are highlighted in yellow for VH and in blue for VL. (B) SDS-PAGE analysis of scFv-LT recombinant antibody expression and purification. Lanes: 1. C43(DE3) non-induced fraction; 2. C43(DE3) induced fraction; 3. Insoluble fraction; 4. Purified scFv-LT.
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pone.0131484.g001: Construction, expression and purification of scFv-LT.(A) scFv-LT amino acids sequence: VH—Heavy chain variable domain, L—Linker and VL—Light chain variable domain sequence. CDRs are highlighted in yellow for VH and in blue for VL. (B) SDS-PAGE analysis of scFv-LT recombinant antibody expression and purification. Lanes: 1. C43(DE3) non-induced fraction; 2. C43(DE3) induced fraction; 3. Insoluble fraction; 4. Purified scFv-LT.

Mentions: DNA fragments of approximately 720 bp were obtained from synthetic genes (VH-linker-VL orientation) amplification corresponding to scFv-LT and scFv-ST. These fragments were subcloned into pET28a vector, confirmed by restriction analysis using BamHI / XhoI, and sequencing, which showed that scFv-LT and scFv-ST fragments presented 100% identities with the corresponding synthetic genes, and the complementary domain regions (CDRs) sequences were preserved (Figs 1A and 2A).


Single Chain Variable Fragments Produced in Escherichia coli against Heat-Labile and Heat-Stable Toxins from Enterotoxigenic E. coli.

Ozaki CY, Silveira CR, Andrade FB, Nepomuceno R, Silva A, Munhoz DD, Yamamoto BB, Luz D, Abreu PA, Horton DS, Elias WP, Ramos OH, Piazza RM - PLoS ONE (2015)

Construction, expression and purification of scFv-LT.(A) scFv-LT amino acids sequence: VH—Heavy chain variable domain, L—Linker and VL—Light chain variable domain sequence. CDRs are highlighted in yellow for VH and in blue for VL. (B) SDS-PAGE analysis of scFv-LT recombinant antibody expression and purification. Lanes: 1. C43(DE3) non-induced fraction; 2. C43(DE3) induced fraction; 3. Insoluble fraction; 4. Purified scFv-LT.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496030&req=5

pone.0131484.g001: Construction, expression and purification of scFv-LT.(A) scFv-LT amino acids sequence: VH—Heavy chain variable domain, L—Linker and VL—Light chain variable domain sequence. CDRs are highlighted in yellow for VH and in blue for VL. (B) SDS-PAGE analysis of scFv-LT recombinant antibody expression and purification. Lanes: 1. C43(DE3) non-induced fraction; 2. C43(DE3) induced fraction; 3. Insoluble fraction; 4. Purified scFv-LT.
Mentions: DNA fragments of approximately 720 bp were obtained from synthetic genes (VH-linker-VL orientation) amplification corresponding to scFv-LT and scFv-ST. These fragments were subcloned into pET28a vector, confirmed by restriction analysis using BamHI / XhoI, and sequencing, which showed that scFv-LT and scFv-ST fragments presented 100% identities with the corresponding synthetic genes, and the complementary domain regions (CDRs) sequences were preserved (Figs 1A and 2A).

Bottom Line: These strains can produce two enterotoxins associated with the manifestation of clinical symptoms that can be used to detect these pathogens.Both antibody fragments were able to recognize the cell-interacting toxins by immunofluorescence, the purified toxins by ELISA and also LT-, ST- and LT/ST-producing ETEC strains.The developed recombinant scFvs against LT and ST constitute promising starting point for simple and cost-effective ETEC diagnosis.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Bacteriologia, Instituto Butantan, São Paulo, SP, Brasil.

ABSTRACT

Background: Diarrhea is a prevalent pathological condition frequently associated to the colonization of the small intestine by enterotoxigenic Escherichia coli (ETEC) strains, known to be endemic in developing countries. These strains can produce two enterotoxins associated with the manifestation of clinical symptoms that can be used to detect these pathogens. Although several detection tests have been developed, minimally equipped laboratories are still in need of simple and cost-effective methods. With the aim to contribute to the development of such diagnostic approaches, we describe here two mouse hybridoma-derived single chain fragment variable (scFv) that were produced in E. coli against enterotoxins of ETEC strains.

Methods and findings: Recombinant scFv were developed against ETEC heat-labile toxin (LT) and heat-stable toxin (ST), from previously isolated hybridoma clones. This work reports their design, construction, molecular and functional characterization against LT and ST toxins. Both antibody fragments were able to recognize the cell-interacting toxins by immunofluorescence, the purified toxins by ELISA and also LT-, ST- and LT/ST-producing ETEC strains.

Conclusion: The developed recombinant scFvs against LT and ST constitute promising starting point for simple and cost-effective ETEC diagnosis.

No MeSH data available.


Related in: MedlinePlus