Journal Article

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

Munhoz, Danielle D., Luz, Daniela, Ramos, Oscar H. P., Andrade, Fernanda B., Nepomuceno, Roberto, Ozaki, Christiane Y., Yamamoto, Bruno B., Piazza, Roxane M. F., Elias, Waldir P., Silveira, Caio R. F., Abreu, Patrícia A. E., Horton, Denise S. P. Q., Silva, Anderson

2015

Overview

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

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Amino Acid Sequence / Antigens / Bacterial Toxins - immunology / Chains / Colonization / Developing countries / Diagnostic systems / Diarrhea / E coli / Enterotoxigenic Escherichia coli - isolation & purification / Enterotoxigenic Escherichia coli - metabolism / Enterotoxins / Enterotoxins - immunology / Enzyme-linked immunosorbent assay / Escherichia coli / Escherichia coli Proteins - immunology / Fragmentation / Fragments / Genetic engineering / Heat / Immunofluorescence / Immunoglobulins / Laboratories / LDCs / Molecular Sequence Data / Polypeptides / Protein expression / Proteins / Recombinant Proteins - isolation & purification / RNA polymerase / Single-Chain Antibodies - biosynthesis / Single-Chain Antibodies - chemistry / Small intestine / Thermal stability / Toxins