2013 Annual Report
1a.Objectives (from AD-416):
The objective of this cooperative research project is to develop an electrochemiluminescence assay for BoNT in foods, using novel monoclonal antibodies developed by our ARS laboratory. Specifically, BoNT occurs in many serotype variations. This Agreement addresses BoNT serotypes A, B, and E. The foods covered under this Agreement include ground beef, liquid eggs, milk, and leafy greens, such as lettuce and spinach.
1b.Approach (from AD-416):
We will test various monoclonal antibodies developed by ARS, as well as additional monoclonal and polyclonal antibodies available through commercial license. Antibodies will be employed in pairs, in sandwich format, using the Meso Scale platform for electrochemiluminescence. For validation, crude supernatant from cultures of each serotype will be spiked into foods, and the Lower Limit Of Detection (LLOD) determined. Formerly 5325-42000-043-10N (4/11).
This is the final report for this project. Immunoassay methods were developed that utilize electrochemiluminescence (ECL) for detection of foodborne toxins. An ECL instrument (Sector 2400 Imager) was loaned by the cooperator until December 2012. Assays for serotypes A and B of botulinum neurotoxin (BoNT), responsible for foodborne botulism were developed and documented: Detection of botulinum neurotoxin serotypes A and B using a chemiluminescent versus electrochemiluminescent immunoassay in food and serum,” J. Agric. Food Chem. 61:755-760 (2013). Previously developed enzyme-linked immunosorbent assay (ELISA) methods for detection of ricin (the toxin from castor bean) were also ported to the ECL platform and documented in several publications that described assays in milk, liquid eggs, and ground beef. The detection of both toxins was better in the ECL platform than with the ELISA platform in most of the food matrices that were tested, with lower matrix interference and somewhat greater sensitivity. The ECL platform entailed higher costs for assay plates than ELISA, but the 2 – 3 fold smaller sample size lowered reagent use. Research progress reported addresses objectives 1: "Develop new assays for bacterial toxins and their variants, using immunological and other methods, with emphasis on applicability to practical problems facing the food industry and regulatory agencies" and 2: "Calibrate in vitro methodology against established animal bioassays, and develop new data on the bioavailability of toxins, the impact of food processing on toxin activities, and the significance of antibody-mediated clearance on toxicity, especially via the oral route of intoxication" of the parent project.