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A High-Throughput Antibody-Based Microarray Typing Platform
Molecular Characterization of Foodborne Pathogens Research Unit, United States Department of Agriculture-North Atlantic Area-Agricultural Research Service-Eastern Regional Research Center, Wyndmoor, PA 19038, USA
NanoDetection Technology, Inc., Franklin, OH 45005, USA
Reference Center, Department of Veterinary and Biomedical Sciences, Pennsylvania State University, University Park, PA 16802, USA
Department of Food Science and Technology, University of Tennessee, Knoxville, TN 37916, USA
Department of Animal Science, University of Tennessee, Knoxville, TN 37916, USA
* Author to whom correspondence should be addressed.
Received: 22 February 2013; in revised form: 9 April 2013 / Accepted: 15 April 2013 / Published: 3 May 2013
Abstract: Many rapid methods have been developed for screening foods for the presence of pathogenic microorganisms. Rapid methods that have the additional ability to identify microorganisms via multiplexed immunological recognition have the potential for classification or typing of microbial contaminants thus facilitating epidemiological investigations that aim to identify outbreaks and trace back the contamination to its source. This manuscript introduces a novel, high throughput typing platform that employs microarrayed multiwell plate substrates and laser-induced fluorescence of the nucleic acid intercalating dye/stain SYBR Gold for detection of antibody-captured bacteria. The aim of this study was to use this platform for comparison of different sets of antibodies raised against the same pathogens as well as demonstrate its potential effectiveness for serotyping. To that end, two sets of antibodies raised against each of the “Big Six” non-O157 Shiga toxin-producing E. coli (STEC) as well as E. coli O157:H7 were array-printed into microtiter plates, and serial dilutions of the bacteria were added and subsequently detected. Though antibody specificity was not sufficient for the development of an STEC serotyping method, the STEC antibody sets performed reasonably well exhibiting that specificity increased at lower capture antibody concentrations or, conversely, at lower bacterial target concentrations. The favorable results indicated that with sufficiently selective and ideally concentrated sets of biorecognition elements (e.g., antibodies or aptamers), this high-throughput platform can be used to rapidly type microbial isolates derived from food samples within ca. 80 min of total assay time. It can also potentially be used to detect the pathogens from food enrichments and at least serve as a platform for testing antibodies.
Keywords: antibody; microarray; bacteria; fluorescence; microtiter plate; typing
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Gehring, A.; Barnett, C.; Chu, T.; DebRoy, C.; D'Souza, D.; Eaker, S.; Fratamico, P.; Gillespie, B.; Hegde, N.; Jones, K.; Lin, J.; Oliver, S.; Paoli, G.; Perera, A.; Uknalis, J. A High-Throughput Antibody-Based Microarray Typing Platform. Sensors 2013, 13, 5737-5748.
Gehring A, Barnett C, Chu T, DebRoy C, D'Souza D, Eaker S, Fratamico P, Gillespie B, Hegde N, Jones K, Lin J, Oliver S, Paoli G, Perera A, Uknalis J. A High-Throughput Antibody-Based Microarray Typing Platform. Sensors. 2013; 13(5):5737-5748.
Gehring, Andrew; Barnett, Charles; Chu, Ted; DebRoy, Chitrita; D'Souza, Doris; Eaker, Shannon; Fratamico, Pina; Gillespie, Barbara; Hegde, Narasimha; Jones, Kevin; Lin, Jun; Oliver, Stephen; Paoli, George; Perera, Ashan; Uknalis, Joseph. 2013. "A High-Throughput Antibody-Based Microarray Typing Platform." Sensors 13, no. 5: 5737-5748.