Yaron Hillman  ^_ The Shmunis School of Biomedicine and Cancer Research, George S. Wise Faculty of Life Sciences, Green building, Tel-Aviv University, Tel Aviv 6997801, Israel.
Jenia Gershberg  ^_ Department of Microbiology, Immunology and Genetics, Faculty of Health Sciences, Ben-Gurion University of the Negev, P.O. Box 653, Beer Sheva 8410501, Israel.
Dan Lustiger  ^_ The Shmunis School of Biomedicine and Cancer Research, George S. Wise Faculty of Life Sciences, Green building, Tel-Aviv University, Tel Aviv 6997801, Israel.
Dan Even  ^_ The Shmunis School of Biomedicine and Cancer Research, George S. Wise Faculty of Life Sciences, Green building, Tel-Aviv University, Tel Aviv 6997801, Israel.
Dor Braverman  ^_ Department of Microbiology, Immunology and Genetics, Faculty of Health Sciences, Ben-Gurion University of the Negev, P.O. Box 653, Beer Sheva 8410501, Israel.
Yael Dror  ^_ The Shmunis School of Biomedicine and Cancer Research, George S. Wise Faculty of Life Sciences, Green building, Tel-Aviv University, Tel Aviv 6997801, Israel.³
Sefi Vernick  ^_ Institute of Agricultural Engineering, Agricultural Research Organization, Volcani Center, 68 Hamaccabim Rd, Rishon Lezion 5025001, Israel.
Neta Sal-Man  ^_ Department of Microbiology, Immunology and Genetics, Faculty of Health Sciences, Ben-Gurion University of the Negev, P.O. Box 653, Beer Sheva 8410501, Israel.
Yariv Wine  ^_ The Shmunis School of Biomedicine and Cancer Research, George S. Wise Faculty of Life Sciences, Green building, Tel-Aviv University, Tel Aviv 6997801, Israel.

It is predicted that the antibiotic resistance crisis will result in an annual death rate of 10 million people by the year 2050. To grapple with the challenges of the impending crisis, there is an urgent need for novel and rapid diagnostic tools. In this study, we developed a novel monoclonal antibody-named mAb-EspB-B7-that targets the EspB protein, a component within the bacterial type 3 secretion system (T3SS), which is mainly expressed in Gram-negative pathogens and is essential for bacterial infectivity. We found that mAb-EspB-B7 has high affinity and specificity toward recombinant and native EspB proteins; is stable over a range of pH levels, temperatures, and salt concentrations; and retains its functionality in human serum. We identified the epitope for mAb-EspB-B7 and validated it by competitive enzyme-linked immunosorbent assay (ELISA). Since this epitope is conserved across several T3SS-harboring pathogens, mAb-EspB-B7 holds great potential for development as an active component in precise and rapid diagnostic tools that can differentiate between commensal and pathogenic bacterial strains. To this end, we integrated the well-characterized monoclonal antibody into an electrochemical biosensor and demonstrated its high specificity and sensitivity capabilities in detecting pathogenic bacterial T3SS-associated antigens as well as intact bacteria. We foresee that in the near future it will be possible to design and develop a point-of-care biosensor with multiplexing capabilities for the detection of a panel of pathogenic bacteria.