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Escherichia coli O157:H7 is a serious human pathogen that causes hemorrhagic colitis, and occasionally hemolytic uremic syndrome. Identification of the O157 antigen is an essential part of the detection and management of E. coli O157:H7. A quadroma P126 secreting a bispecific hybrid MAb (bsMAb), which recognizes both E. coli O157 and horseradish peroxidase in one molecule was produced by somatic hybridization of hybridomas specific for E. coli O157 and HRPO molecule. A bridge ELISA was used to select the quadromas obtained for bispecific monoclonal antibody purification and characterization. Benzhydroxamic-acid agarose (BHA) affinity co-chromatography was used as a convenient one-step method for purifying the HRPO-bsMAb complex for ultrasensitive diagnostic applications. Sandwich ELISA for detecting E. coli O157:H7 with HRPO-bsMAb allows quick one step detection of spiked E. coli O157:H7. The detection sensitivities were 100 CFU, 750 CFU and 500 CFU per 1 ml of tap water, lake water and apple juice respectively by microtiter assay. E. coli O157:H7 detection with immunofilter ELISA and immunomagnetic ELISA formats was approximately 1 CFU/ml and 10 CFU/ml respectively. BsMAbs avoid enzyme conjugation, has highest specific activity and molecular uniformity without aggregates and contribute to good signal to noise ratios. This new bispecific antibody can be generated and purified from quadroma cultures by affinity co-chromatography in one step and can be used to develop a new generation of assays for public health applications in water, food and human sample testing.