|Andreotti, Peter - PERKIN-ELMER, MD|
|Venkateswaran, Kodumudi - UC, LLNL, LIVERMORE, CA|
|Mcbride, Mary - UC, LLNL, LIVERMORE, CA|
|USDA Personnel, - USDA, ALBANY, CA|
Submitted to: United States Japan Natural Resources Protein Panel
Publication Type: Proceedings
Publication Acceptance Date: September 14, 2001
Publication Date: October 15, 2001
Citation: Stanker, L.H., Sheffield, C., Beier, R.C. Andreotti, P., Venkateswaran, K., McBride, M.T., Ravva, S.V., Duffy, B.K., Mandrell, R.E. 2001. Bacterial Detection Using Sensitive Immunological Methods. In Proceedings of the 30th United States-Japan Cooperative Program in Natural Resources (UJNR) Protein Resources Panel, October 15-19, 2001, Tsukuba, Ibaraki, Japan. p. 33-40. Interpretive Summary: Detecting human pathogenic bacteria in complex natural environments such as food or manure requires multiple approaches. This study describes the characterization of immunological methods using a novel set of monoclonal antibodies for the specific detection of a particularly troublesome pathogen, Campylobacter. These antibodies enable the rapid detection and the differentiation of 3 different species of the pathogen. In addition, broadly cross-reactive monoclonal antibodies also allow identification of Campylobacter species in general. A new technique called time-resolved fluorescence detection, which utilizes rare earth elements such as europium conjugated to the antibodies, was incorporated to greatly enhance the sensitivity of our assay compared to previously described methods. We also describe the application of our antibodies in a 14-plex immunoassay that utilizes fluorescence detection on microspheres in a flow analyzer.
Technical Abstract: Detection of pathogenic bacteria in complex biological matrices such as food or manure is a challenge requiring multiple approaches. In this study, a set of monoclonal antibodies that bind to different species of Campylobacter are further characterized and they are applied in different multiplexed immunoassays. These monoclonal antibodies allow for rapid detection and species identification of C. coli, C. jejuni and C. lari. I addition, broadly cross-reactive monoclonal antibodies also allow identification of other Campylobacter species. Using Europium conjugated anti-Campylobacter antibodies, and a microfiltration format, time-resolved fluorescence detection was incorporated into an ultra-sensitive immunoassay. The assay was able to detect as few as 10 CFU/mL of C. lari and had a linear response over several logs of bacterial concentration. In contrast, traditional ELISA formats have sensitivities of approximately 10 5 CFU/mL and usually are only linear over 1 to 1.5 logs of bacterial concentration. Finally data is presented demonstrating the application of a 14-plex immunoassay that utilizes fluorescence detection on microspheres in a flow analyzer.