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Aims:  To study the reaction patterns of selected antibodies to Escherichia coli O157:H7, Salmonella enterica serotype Enteritidis and Listeria monocytogenes cells exposed to various environmental stresses. Methods and Results:  Escherichia coli O157:H7, Salmonella Enteritidis and L. monocytogenes cells subjected to different environmental stress of temperatures (4 and 45°C), NaCl (5·5%), oxidative stress (15 mmol−1 H2O2), acidic pH (5·5) and ethanol (5%) for 3 h (short‐term stress) or for 5 days (long‐term stress) were analysed by ELISA and Western blotting. The ELISA results indicated that most stresses caused 12–16% reductions in reaction for anti‐E. coli O157:H7 and 20–48% reductions for anti‐Salmonella polyclonal antibodies during short‐term stress, whereas the most stresses exhibited enhanced reaction (44–100% increase) with the anti‐L. monocytogenes polyclonal antibody. During long‐term stress exposure to combined stress conditions of pH 5·5, 3·5% NaCl at 12°C or at 4°C, antibody reactions to the three pathogens were highly variable with the combined stress at 4°C showing the most reductions (8–40%). Likewise, there were about 18–59% reductions in antibody reactions with pathogens when cultured in hotdog samples with the combined stress conditions. Western blot analyses of crude cell surface antigens from both short‐ and long‐term stressed cells revealed that the changes in antibody reactions observed in ELISA were either because of repression, expression or possible denaturation of antigens on the surface of cells. Conclusions:  Overall, the antibody reactions were significantly reduced in pathogens exposed to both short‐ and long‐term environmental stresses in culture medium or in meat sample because of expression, repression or denaturation of specific antigens in cells. Significance and Impact of the Study:  In order to ensure the reliable detection of foodborne pathogens using antibody‐based methods, the influence of stress on antibody reactions should be thoroughly examined and understood first as the physiological activities in cells are often altered in response to a stress.

Selective enrichment broths are frequently used to recover stressed Listeria cells to detectable levels, but the ability of antibodies to detect these cells from various commonly used enrichment media is unknown. In this study, a polyclonal (PAb) and monoclonal (MAb) antibody were used to examine the variation in antigen expression on healthy or stress-recovered Listeria monocytogenes cells grown in brain heart infusion broth, buffered Listeria enrichment broth (BLEB), Listeria repair broth (LRB), University of Vermont medium (UVM), and Fraser broth (FB) for immunodetection. Indirect enzyme-linked immunosorbent assay (ELISA) data showed that L. monocytogenes subjected to stresses (acid, cold, heat, and salt) and then grown in BLEB gave the highest reaction with the anti-Listeria PAb while those grown in LRB gave the highest reaction with the MAb C11E9. Cells grown in UVM and FB gave poor ELISA values with both antibodies. Western blotting with PAb revealed differential expression of surface proteins of 62, 58, 50, 43, and 30 kDa on L. monocytogenes cells, with most proteins displaying elevated expression in BLEB and LRB but reduced or no expression in UVM or FB. Similar differential expressions were noticed for C11E9. PAb-reactive proteins were identified as putative LPXTG-motif cell-wall anchor-domain protein (62 kDa; lmo0610), flavocytochrome C fumarate reductase chain A homolog protein (58 kDa; lmo0355), enolase (50 kDa; lmo2455), glyceraldehyde 3-phosphate dehydrogenase (43 kDa; lmo2459), and hypothetical phospho-sugar binding protein (30 kDa; lmo0041), respectively, and the MAb-reactive 66-kDa protein was confirmed to be N-acetylmuramidase (lmo2691). In conclusion, BLEB and LRB favorably supported increased expression of antigens and proved to be superior to UVM and FB for immunodetection of stressed L. monocytogenes cells.

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Recent outbreaks of food borne illnesses continue to support the need for rapid and sensitive methods for detection of foodborne pathogens. A method for detecting Listeria monocytogenes in food samples was developed using an automated fiber-optic-based immunosensor, RAPTOR ™. Detection of L. monocytogenes in phosphate buffered saline (PBS) was performed to evaluate both static and flow through antibody immobilization methods for capture antibodies in a sandwich assay. Subsequent detection in frankfurter samples was conducted using a flow through immobilization system. A two stage blocking using biotinylated bovine serum albumin (b-BSA) and BSA was effectively employed to reduce the non-specific binding. The sandwich assay using static or flow through mode of antibody immobilization could detect 1×10 3 cfu/ml in PBS. However, the effective disassociation constant K d and the binding valences for static modes of antibody immobilization in spiked PBS samples was 4×10 5 cfu/ml and 4.9 as compared to 7×10 4 cfu/ml and 3.9 for flow through method of antibody immobilization. Thus the sensitive flow-through immobilization method was used to test food samples, which could detect 5×10 5 cfu/ml of L. monocytogenes in frankfurter sample. The responses at the lowest detectable cell numbers in the frankfurter samples was 92.5 ± 14.6 pA for L. monocytogenes to comparative responses of 27.9 ± 12.2 and 31 ± 14.04 pA obtained from Enterococcus Sensors 2006, 6 809 faecalis and Lactobacillus rhamnosus (control species), respectively. The effective K d and binding valency from spiked frankfurter samples was 4.8×10 5 cfu/ml and 3.1, thus showing highly sensitive detection can be achieved using the RAPTOR ™ biosensor even in the presence of other bacterial species in the matrix.