"We developed a sandwich immunofluorescence assay in a microarray format for capturing and detecting L. monocytogenes, E. coli O157:H7, and S. Enteritidis..."

Multipathogen screening using immunomicroarry

Investigator: Arun K. Bhunia (Department of Food Science)

Project Report 2008 - 2009

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Project Rationale

Most assay methods for detecting pathogenic foodborne bacteria are developed for the detection of a single target pathogen. As a result, these methods can be costly when testing for multiple pathogens from a single sample because separate assay methods are required. The overall objective of this project was to develop a 96-well plate-based sensitive immunoassay for concurrent detection of three pathogens?Listeria monocytogenes, Escherichia coli O157:H7 and Salmonella Enteritidis?occurring simultaneously in a test sample. Multi-pathogen analysis on a single assay platform will eliminate the need for separate tests for multiple microorganisms, reduce test costs, and ultimately improve food safety.

Project Objectives

  • Develop a microarray assay in a 96-well plate and glass slide using a sandwich immunoassay for three pathogens.
  • Optimize growth and enrichment of three pathogens (healthy or stressed) spiked in model food samples in a selective enrichment broth for use with the microarray.
  • Evaluate the performance of the Pathogen Enrichment Device.

Project Highlights

We developed a sandwich immunofluorescence assay in a microarray format for capturing and detecting L. monocytogenes, E. coli O157:H7, and S. Enteritidis using antibodies developed in our laboratory. The different antibodies for each pathogen were screened for their efficiency either as capture or detection antibodies. Streptavidin-coated microtiter plates were used to immobilize biotinylated antibodies for the detection of target pathogens. For L. monocytogenes, we used biotinylated-P66 as a capture antibody, and horseradish peroxidase or Alexa Fluor (AF)-labeled C11E9 as a detection antibody. We used biotinylated-anti-E. coli polyclonal Ab and AF-labeled-pET32a antibodies for E. coli, and biotinylated-anti-Salmonella PAb and AF-labeled MAb-2F11 antibodies for S. Enteritidis. These antibody combinations were applied to a sandwich fluorescent immunoassay in 96-well plates. The detection limit was determined to be 105 cells/well. We have demonstrated that this 96-well microplate-based immunoassay can be used for specific detection of L. monocytogenes, E. coli O157:H7, and S. Enteritidis when grown in ready-to-eat meat products such as beef, chicken, and turkey.