An antibody microarray,in multiwell plate format,for multiplex screening of foodborne pathogenic bacteria and biomolecules |
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Authors: | Andrew G Gehring David M Albin Sue A Reed Shu-I Tu Jeffrey D Brewster |
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Institution: | (1) Microbial Biophysics and Residue Chemistry Research Unit, United States Department of Agriculture–Agricultural Research Service, Eastern Regional Research Center, 600 East Mermaid Lane, Wyndmoor, PA 19038, USA |
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Abstract: | Intoxication and infection caused by foodborne pathogens are important problems worldwide, and screening tests for multiple
pathogens are needed because foods may be contaminated with multiple pathogens and/or toxic metabolites. We developed a 96-well
microplate, multiplex antibody microarray method to simultaneously 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) in a single sample.
Microarrayed spots of capture antibodies against the targeted analytes were printed within individual wells of streptavidin-coated
polystyrene 96-multiwell microtiter plates and a sandwich assay with fluorescein- or Cy3-labeled reporter antibodies was used
for detection. (Printing was achieved with a conventional microarray printing robot that was operated with custom-developed
microplate arraying software.) Detection of the IgG was realized from ca. 5 to 25 ng/mL, and detection of E. coli O157:H7 and S. typhimurium was realized from ca. 106 to 109 and ca. 107 to 109 cells/mL, respectively. Multiplex detection of the two bacteria and the IgG in buffer and in culture-enriched ground beef
filtrate was established with a total assay (including detection) time of ca. 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
a small decrease in response was observed for E. coli O157:H7. The multiwell plate, multiplex antibody microarray platform developed here demonstrates a powerful approach for
high-throughput screening of large numbers of food samples for multiple pathogens and toxins. |
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Keywords: | Antibody microarray Bacteria Fluorescence Immunoassay Multiwell microtiter plate Multiplex |
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