2012 Annual Report 1a.Objectives (from AD-416): The objective of this cooperative research project is to develop a multiplex superparamagnetic fluorescent microbead immunochemical assay for foodborne pathogens in food. Specifically, the scope of this project includes simultaneous detection and identification of the seven most common strains of Shiga toxin-producing Escherichia coli (STEC) based on O antigen: O157, O26, O111, O103, O121, O45, and O145. It also includes Listeria monocytogenes serotypes 1/2a, 1/2b, and 4b; and Salmonella enterica serovar Typhimurium. Foods of interest include ground beef, liquid egg, milk, and leafy greens. Performance characteristics of the product require sensitivity of at least one infectious dose of live bacteria per serving of food. 1b.Approach (from AD-416): The overall design includes three components: (1) cultural enrichment, (2) sandwich ELISA format assay using monoclonal antibodies, and (3) a microbead array platform. The improvements to the cultural enrichment step will be accomplished using incremental adjustments of existing standard microbiological media and do not concern this Agreement. An enrichment step is necessary to provide sufficient sensitivity for an assay that must detect the presence of only a few organisms in a sample of several hundred grams of food. The sandwich ELISA format we will employ is commonly performed in microplate format, and is typical for microbead immunochemical assays developed for the Luminex platform. We will use monoclonal and polyclonal antibodies provided by SDIX. Each antigen assay will be deployed on a unique fluorescent labeled microbead set. Specificity and sensitivity will be confirmed using bacteria grown in culture broth. Validation will be performed by University of South Florida (USF) and FDA. For validation experiments pathogens will be spiked into foods, and the Lower Limit Of Detection will be determined for each organism, in the presence of other pathogens and typical background flora. 3.Progress Report: Currently, we are finishing up 2 manuscripts detailing development of a 9-plex microbead assay for all of the 7O-antigens (O157, O26, O111, O103, O121, O45, and O145) and the two Shigella toxins (Stx 1, Stx2). The first paper describes validation of the assay against 160 field isolates of STEC. The second one, describes a comparison of our 9-plex immunoassay against a molecular (PCR-based) assay developed by Cooperators at FDA. Research progress reported, addresses objective 1 of the parent project.