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

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.

Fig 1. Construction, expression and purification of scFv-LT.

(A) scFv-LT amino acids sequence: V_H—Heavy chain variable domain, L—Linker and V_L—Light chain variable domain sequence. CDRs are highlighted in yellow for V_H and in blue for V_L. (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.

Fig 2. Construction, expression and purification of scFv-ST.

(A) scFv-ST amino acids sequence: V_H—Heavy chain variable domain, L—Linker and V_L—Light chain variable domain sequences. CDRs are highlighted in yellow for V_H and in blue for V_L. (B) SDS-PAGE analysis of scFv-ST recombinant antibody expression and purification. Lanes: 1. BL21(DE3) non-induced fraction; 2. BL21(DE3) induced fraction; 3. Insoluble fraction; 4. Purified scFv-ST.

Fig 3. Reactivity of scFv-LT recombinant antibody against LT toxin.

(A) Nitrocellulose membrane containing the LT toxin protein after SDS-PAGE 15% were subjected to immunoblotting with antibodies. Lanes: 1. scFv-LT 200 μg/mL, 2. 50 μg/mL anti-LT mAb. (B) Immunofluorescence assay after LT interaction with Y-1 cells. Panel 1. Reactivity with scFv-LT; Panel 2. Reactivity with anti-LT mAb. Panel 3. Reactivity with scFv-LT in the absence of LT toxin. Panel 4. Light microscopy. (C) scFv-LT titration with LT toxin by capture ELISA. (D) Detection of LT in culture supernatants of LT- and LT/ST-producing ETEC isolates by capture ELISA.

Fig 4. Reactivity of scFv-ST recombinant antibody against ST toxin.

(A) PVDF membranes containing the bacterial lysates fractions after PAGE 10%/tricine gel was subjected to immunoblotting with antibodies. 1. 30 μg/mL of scFv-ST. 2. 30 μg/mL anti-ST mAb. (B) Immunofluorescence assay after ST interaction with Caco-2 cells. Panel 1. Reactivity with scFv-ST; Panel 2. Reactivity with anti-ST mAb. Panel 3. Reactivity with scFv-ST in the absence of ST toxin. Panel 4. Light microscopy. (C) scFv-ST titration with ST toxin by indirect ELISA. (D) Detection of ST in culture supernatants of ST- and LT/ST-producing ETEC isolates by indirect ELISA.