Aim: To evaluate the potential of an intergeneric multidomain recombinant chimeric protein for the simultaneous detection of Bacillus anthracis, Yersinia pestis and Staphylococcal enterotoxin B. Methods and Results: Truncated portions of protective antigen (pag) of B.anthracis, fraction 1 capsular antigen (F1) of Y.pestis and enterotoxin B (entB) of Staphylococcus aureus were PCR amplified and linked each other using ligation-dependent cloning. The fusion gene was codon-optimized for expression in Escherichia coli and encoded a 55kDa recombinant PFE protein (rPFE). Hyperimmune antiserum raised against rPFE specifically reacted individually with the native PA of B.anthracis, F1 antigen of Y.pestis and SEB of S.aureus on Western blot analysis as well as in enzyme-linked immunosorbent assay (ELISA). For simultaneous detection of these three antigens from culture supernatants, common media consisting of BHI broth supplemented with 0 center dot 2% xylose were used. To assess the detection capability, a known number of these organisms (10(8)-10(2) CFUml(-1)) were experimentally spiked on to the meat and blood samples, the polyclonal antibodies were again clearly able to identify all three target proteins up to a dilution of 10(5) CFUml(-1). Conclusions: This recombinant chimeric protein-based immunodetection approach may eventually provide advantages over PCR formats during onsite investigations of biological emergencies or even during routine testing by laboratories. Significance and Impact of the Study: The trivalent recombinant PFE protein could be a novel intervention for possible diagnosis/detection of potential biological agents simultaneously in environmental and clinical samples to reduce the responding time and minimize the impact of the bioattack.