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

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. 10(6) to 10(9) and ca. 10(7) to 10(9) 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.

Detection of Enterobacter sakazakii and Other Pathogens Associated with Infant Formula Powder by Use of a DNA Microarray

“…Recently, various reports have demonstrated the efficacy of this approach in the detection of waterborne pathogens (21), marine fish pathogens (11), and other food-borne pathogens (7,10,19,28,30). To our knowledge, this study describes for the first time the application of DNA microarray technology for the rapid and reliable detection of pathogens associated with PIF contamination.…”

Recent developments in rapid multiplexed bioanalytical methods for foodborne pathogenic bacteria detection

“…Microtiter plate-based planar microarrays have been developed with the aim to facilitate their laboratory implementation, being their format adaptable to automated 96-well microtiter plate handling instrumentation (e.g., robotic dispensers, plate washers, incubators) [6,[42][43][44]. Recently, we designed a 96-well microtiter plate, in which each well is internally divided into four subwells, thus allowing separate immobilization of four distinct antibodies in the bottom of each well.…”

“…Employing this system, a fluorescence multiplex sandwich immunoassay was developed for simultaneous detection of E. coli O157:H7 and S. typhimurium in cultureenriched ground beef filtrate down to 10 6 -10 7 cells mL −1 and 2.5 h total assay time [44]. The technology developed by Meso Scale Diagnostics, LLC (Gaithersburg, MD, http:// www.mesoscale.com) also allows the development of microtiter plate-based multiplexed immunoassays employing electrochemiluminescence (ECL) detection.…”

Pattern recognition of shape-encoded hydrogel biosensor arrays

“…Recent advances in biotechnology have given researchers the ability to perform rapid identification of DNA sequences, 1-8 proteins, [9][10][11][12][13] and small molecules 14-16 on feature-dense biochip sensors. Whether their function is to diagnose disease, detect specific DNA sequences, screen for bioterrorism agents, or identify specific cancer variants, biochips of all types are ushering in a new era of medical and biological research.…”