Design of ELISA-based diagnostic system for detection of enterohaemorrhagic Escherichia coli

Abstract

Background and objectives: Escherichia coli (E. coli) O157:H7 is an intestinal pathogen of humans and animals, which causes serious gastrointestinal, urinary tract infection and hemolytic uremic syndrome. Connecting to the host cell is important in pathogenesis. EspA, Intimin and Tir proteins (EIT) are the most important bacterial features in the process of binding. These antigens can be very useful in detecting these bacteria. The aim of this study was to produce recombinant EspA, Intimin and Tir proteins (rEIT) to detect pathogenic E. coli O157:H7 by means of ELISA method.

Materials and methods: The eit recombinant gene was expressed using IPTG in E. coli BL21 (DE3) and evaluated by western blotting. The purified rEIT protein was injected to rabbits and mice subcutaneously. Purified antibody was evaluated using indirect, competitive and sandwich ELISA confirming the precise detection of E. coli O157: H7.

Results: Indirect, competitive and sandwich ELISA specifically detected E. coli O157:H7 and each methods had the ability to identify more than 10⁴, 10⁴, 10³ bacteria. The specificity of this method was evaluated by Entroheamoragic E. coli, enterotoxygenic E. coli, Klebsiella pneumoniae, Vibrio cholera and Acinetobacter.

Conclusion: These methods are the fastest, most accurate and cost effective methods for diagnosis of E. coli O157: H7, comparing to the conventional methods.

Keywords: Competitive ELISA; Escherichia coli O157:H7; Indirect enzyme-linked immunosorbent assay (ELISA); Sandwich ELISA.

Fig. 1.

Expression, purification and western blot analysis of recombinant EIT. A) Electrophoresis of the recombinant EIT protein: 1) Protein size marker, 2) Induced sample without IPTG, 3) Sample after induction (supernatant), 4) Sample after induction resulted from deposition (inclusion body). B) Purified recombinant protein with Ni-NTA column. 1) Protein size marker, 2) Extract from the lysate bacteria, 3) Flow through, 4) Washing buffer with 30 mM imidazole (pH:8), 5–8) Elution buffer with 300 mM imidazole and 5% glycerol, 9) MES buffer (20 mM). C) The confirmation of EIT recombinant protein by Western blotting product. 1) Protein size marker, 2) recombinant protein, 3) Non-induced as control.

Fig. 2.

Determination of antibody titer in mice and rabbits by ELISA method. A) ELISA for determination of antibody titer against recombinant protein in the mouse animal model. B) ELISA for determination of antibody titer against recombinant protein in the rabbit animal model.

Fig. 3.

Purification of antibody from mice and rabbits by G column. A) Purification mouse IgG antibody using G column, 1) Protein weight marker, 2) Flow through, 3) Washing buffer (pH=7.2), 4–8) Elution buffer with 100 mM glycine solution (pH=3). B) Purification rabbit IgG antibody using G column, 1) Flow through, 2) Washing buffer (pH=7.2), 3) Protein weight marker, 4–9) Elution buffer with 100 mM glycine solution (pH=3).

Fig. 4.

ELISA for evaluating purified IgG. A) ELISA for determination of purified IgG titer produced in mouse and rabbit. B) Indirect ELISA using E. coli O157:H7 against with mouse and rabbit antibody. C) Sandwich ELISA using E. coli O157:H7 against mouse and rabbit antibody.

Fig. 5.

Competitive ELISA with EIT antigen and E. coli O157:H7. A) ELISA using EIT antigen with mouse antibody. B) ELISA using E. coli O157:H7 bacteria with mouse antibody.

Fig. 6.

Determination of specificity of ELISA using some bacteria with mouse and rabbit antibodies.

Fig. 7.

Sandwich and indirect ELISA to evaluate clinical samples. A) Sandwich ELISA using clinical samples with rabbit antibody as capture antibody and mouse antibody used for detection. B) Indirect ELISA using clinical samples, positive and negative control with rabbit antibody.