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United States Department of Agriculture

Agricultural Research Service

Research Project: DETECTION AND TYPING OF FOOD-BORNE PATHOGENS

Location: Molecular Characterization of Foodborne Pathogens

Title: High-throughput antibody microarray for bacteria and toxins

Authors
item Gehring, Andrew
item Brewster, Jeffrey
item He, Yiping
item Irwin, Peter
item Paoli, George
item Simons, Tawana
item Tu, Shu-I
item Uknalis, Joseph

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: May 10, 2013
Publication Date: N/A

Technical Abstract: Ingestion of pathogenic bacteria in foods often results in illnesses that are of worldwide concern. Hence, our research efforts have focused on developing screening tests capable of multiplexed detection of foodborne bacteria and associated toxins. In this study, we describe the combination of a sandwich fluorescence immunoassay with a high-throughput, multiwell plate microarray detection platform. Relatively inexpensive polystyrene plates were employed as microarray substrates that were array-printed with passively adsorbed, capture antibodies. Among several sample reaction conditions (e.g., static vs. agitation) that were compared, centrifugation was the only one found to significantly improve localization of bacteria (pathogenic E. coli O157:H7) to planar capture surfaces containing the printed antibodies. Immobilized bacteria were labeled with a fluorescent dye-antibody conjugate to allow for quantitative detection of the antibody captured bacteria using laser-induced fluorescence scanning. Shiga toxin could be simultaneously detected along with the cells, but none of the reaction mixture conditions employed during sample incubation improved detection of the toxin. The optimized assay had demonstrated limits of detection of ca. 10**5 cells/mL and 100 ng/mL for E. coli O157:H7 and Shiga toxin 1, respectively, in a total assay time of ca. 75 min. This new procedure may find use by food producers and testing laboratories for the high-throughput screening of food samples for pathogens.

Last Modified: 10/21/2014
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