Polyclonal Antibody-Based Sandwich ELISA for Detection of Escherichia coli O۱۵۷:H۷

Introduction: Shiga toxin-producing E. coli (STEC) are bacteria causing severe foodborne diseases, with E. coli O۱۵۷:H۷ being a significant public health concern. Infection may occur following exposure to or drinking the contaminated water or the consumption of the contaminated food, especially meat and dairy products. We aimed to optimize a sandwich ELISA method using purified poly-IgG against HI chimera protein for E. coli O۱۵۷:H۷ detection.Materials and Methods: We induced the recombinant chimeric antigen (HI) in a prokaryotic host and purified it through a Ni-NTA column. After refolding the antigen, mice and rabbits were immunized and the poly-IgGs were purified from sera using a protein G column.Results: Recombinant HI (۶۰ kDa) was expressed in E. coli BL۲۱ (yield: ۱.۲ mg/L) and purified via the Ni-NTA column. Antibodies were generated in mice and rabbits, serving as detection and capture antibodies. The optimized antibody concentrations were ۱.۲۵ μg/ml for capture and ۰.۳۱۲ μg/ml for detection. Our sandwich ELISA demonstrated high sensitivity (limit of detection: ۱۰۴ CFU/ml) and specificity for E. coli O۱۵۷:H۷, confirmed by testing against different bacteria.Conclusions: Our developed sandwich ELISA has proven to be a highly sensitive method for the detection of E. coli O۱۵۷:H۷, capable of reliably detecting bacterial concentrations as low as ۱۰۴ CFU/ml.

Keywords:

E. coli O۱۵۷:H۷ , Sandwich ELISA , Recombinant protein , Polyclonal Antibody , Bacterial Detection , Foodborne Infection