Tehran University of Medical Sciences Iranian Journal of Microbiology 2008-3289 17 2 2025 04 05 278 286 Mohammad Javad Rezaei Applied Microbiology Research Center, Biomedicine Technologies Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran Maryam Eidi Department of Genetics and Biotechnology, School of Biological Science, Varamin-Pishva, Branch Islamic Azad University, Varamin, Iran Seyed Ali Mirhosseini Applied Microbiology Research Center, Biomedicine Technologies Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran Rouhollah Kazemi Department of Molecular Biology, Green Gene Company, Tehran, Iran Mohammad Javad Motamedi Department of Molecular Biology, Green Gene Company, Tehran, Iran Soghra Khani Department of Biochemistry, Pasteur Institute of Iran, Tehran, Iran Jafar Amani Applied Microbiology Research Center, Biomedicine Technologies Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran 2024 03 10 2025 03 03 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. https://ijm.tums.ac.ir/index.php/ijm/article/view/4642 https://ijm.tums.ac.ir/index.php/ijm/article/download/4642/1767