An antibody microarray, in multiwell plate format, for multiplex screening of foodborne pathogenic bacteria and biomolecules

Authors: Gehring, Andrew; Albin, David; Reed, Sue; Tu, Shu I; Brewster, Jeffrey

Submitted to: Analytical and Bioanalytical Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/29/2008
Publication Date: 5/1/2008
Citation: Gehring, A.G., Albin, D.M., Reed, S.A., Tu, S., Brewster, J.D. 2008. An antibody microarray, in multiwell plate format, for multiplex screening of foodborne pathogenic bacteria and biomolecules. Analytical and Bioanalytical Chemistry. 391:497-506.

Interpretive Summary: Illness caused by harmful bacteria in foods is an important concern in the United States. The development of screening tests that can detect several different bacteria at once has been investigated. We have developed a 96-multiwell sample plate-based, multiplex antibody microarray method to capture and detect the harmful bacteria Escherichia coli O157:H7 and Salmonella typhimurium, as well as the biomolecule, chicken Immunoglobulin G or IgG antibody (a test substitution for protein-based toxins). We demonstrated that the bacteria were detected by the new technique in pure cultures and also more complex test samples that contained ground beef. A direct association between measured response and amount of the biomolecule was observed from approximately 5 to 25 nanograms per milliliter and in the case of bacteria, the linear range was 10e7 to 10e9 cells per milliliter. Detection of either bacteria was largely unaffected by high amounts of the other bacteria and the IgG, with or without the ground beef present. This new procedure may find use by food producers and testing laboratories for the screening of large numbers of food samples for pathogens and toxins.

Technical Abstract: Intoxication and infection caused by foodborne pathogens are important problems in the United States, and screening tests for multiple pathogen detection have been developed because food producers are known reservoirs of multiple pathogens. We developed a 96-well microplate, multiplex antibody microarray method to capture and detect Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium (S. typhimurium), as well as a biomolecule (chicken immunoglobulin G or IgG) employed as a proteinaceous toxin analog serially diluted in phosphate-buffered saline (PBS) or culture enriched ground beef filtrate. Linear detection of the IgG was realized from ~5 to ~25 ng/mL, beyond which, the fluorescent signal became saturated. Detection of E. coli O157:H7 and S. typhimurium was realized from ~10e6 to 10e9 and ~10e7 to 10e9 cells/mL, respectively. Multiplex detection of the two bacteria and the IgG in ground beef was established using a total assay (including detection) time of ~2.5 h. Detection of S. typhimurium was largely unaffected by high concentrations of the other bacteria and IgG as well as the ground beef filtrate whereas there appeared to be a small decrease in response for E. coli O157:H7. The multiwell plate, multiplex antibody microarray procedure developed here may be useful for screening large number of food samples for pathogens and toxins.