Antibody Microarray for E. coli O157:H7 and Shiga Toxin in Microtiter Plates
Antibody microarray is a powerful analytical technique because of its inherent ability to simultaneously discriminate and measure numerous analytes, therefore making the technique conducive to both the multiplexed detection and identification of bacterial analytes (i.e., whole cells, as well as asso...
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MDPI AG
2015-12-01
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Online Access: | http://www.mdpi.com/1424-8220/15/12/29807 |
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author | Andrew G. Gehring Jeffrey D. Brewster Yiping He Peter L. Irwin George C. Paoli Tawana Simons Shu-I Tu Joseph Uknalis |
author_facet | Andrew G. Gehring Jeffrey D. Brewster Yiping He Peter L. Irwin George C. Paoli Tawana Simons Shu-I Tu Joseph Uknalis |
author_sort | Andrew G. Gehring |
collection | DOAJ |
description | Antibody microarray is a powerful analytical technique because of its inherent ability to simultaneously discriminate and measure numerous analytes, therefore making the technique conducive to both the multiplexed detection and identification of bacterial analytes (i.e., whole cells, as well as associated metabolites and/or toxins). We developed a sandwich fluorescent immunoassay combined with a high-throughput, multiwell plate microarray detection format. Inexpensive polystyrene plates were employed containing passively adsorbed, array-printed capture antibodies. During sample reaction, centrifugation was the only strategy found to significantly improve capture, and hence detection, of bacteria (pathogenic Escherichia coli O157:H7) to planar capture surfaces containing printed antibodies. Whereas several other sample incubation techniques (e.g., static vs. agitation) had minimal effect. Immobilized bacteria were labeled with a red-orange-fluorescent dye (Alexa Fluor 555) conjugated antibody to allow for quantitative detection of the captured bacteria with a laser scanner. Shiga toxin 1 (Stx1) could be simultaneously detected along with the cells, but none of the agitation techniques employed during incubation improved detection of the relatively small biomolecule. Under optimal conditions, the assay had demonstrated limits of detection of ~5.8 × 105 cells/mL and 110 ng/mL for E. coli O157:H7 and Stx1, respectively, in a ~75 min total assay time. |
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language | English |
last_indexed | 2024-04-13T09:19:52Z |
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spelling | doaj.art-6d2c0d38a9444c918292ca83cfecb3c82022-12-22T02:52:38ZengMDPI AGSensors1424-82202015-12-011512304293044210.3390/s151229807s151229807Antibody Microarray for E. coli O157:H7 and Shiga Toxin in Microtiter PlatesAndrew G. Gehring0Jeffrey D. Brewster1Yiping He2Peter L. Irwin3George C. Paoli4Tawana Simons5Shu-I Tu6Joseph Uknalis7Molecular Characterization of Foodborne Pathogens Research Unit, United States Department of Agriculture-Northeast Area, Agricultural Research Service, Eastern Regional Research Center, Wyndmoor, PA 19038, USAMolecular Characterization of Foodborne Pathogens Research Unit, United States Department of Agriculture-Northeast Area, Agricultural Research Service, Eastern Regional Research Center, Wyndmoor, PA 19038, USAMolecular Characterization of Foodborne Pathogens Research Unit, United States Department of Agriculture-Northeast Area, Agricultural Research Service, Eastern Regional Research Center, Wyndmoor, PA 19038, USAMolecular Characterization of Foodborne Pathogens Research Unit, United States Department of Agriculture-Northeast Area, Agricultural Research Service, Eastern Regional Research Center, Wyndmoor, PA 19038, USAMolecular Characterization of Foodborne Pathogens Research Unit, United States Department of Agriculture-Northeast Area, Agricultural Research Service, Eastern Regional Research Center, Wyndmoor, PA 19038, USAMolecular Characterization of Foodborne Pathogens Research Unit, United States Department of Agriculture-Northeast Area, Agricultural Research Service, Eastern Regional Research Center, Wyndmoor, PA 19038, USAMolecular Characterization of Foodborne Pathogens Research Unit, United States Department of Agriculture-Northeast Area, Agricultural Research Service, Eastern Regional Research Center, Wyndmoor, PA 19038, USAMolecular Characterization of Foodborne Pathogens Research Unit, United States Department of Agriculture-Northeast Area, Agricultural Research Service, Eastern Regional Research Center, Wyndmoor, PA 19038, USAAntibody microarray is a powerful analytical technique because of its inherent ability to simultaneously discriminate and measure numerous analytes, therefore making the technique conducive to both the multiplexed detection and identification of bacterial analytes (i.e., whole cells, as well as associated metabolites and/or toxins). We developed a sandwich fluorescent immunoassay combined with a high-throughput, multiwell plate microarray detection format. Inexpensive polystyrene plates were employed containing passively adsorbed, array-printed capture antibodies. During sample reaction, centrifugation was the only strategy found to significantly improve capture, and hence detection, of bacteria (pathogenic Escherichia coli O157:H7) to planar capture surfaces containing printed antibodies. Whereas several other sample incubation techniques (e.g., static vs. agitation) had minimal effect. Immobilized bacteria were labeled with a red-orange-fluorescent dye (Alexa Fluor 555) conjugated antibody to allow for quantitative detection of the captured bacteria with a laser scanner. Shiga toxin 1 (Stx1) could be simultaneously detected along with the cells, but none of the agitation techniques employed during incubation improved detection of the relatively small biomolecule. Under optimal conditions, the assay had demonstrated limits of detection of ~5.8 × 105 cells/mL and 110 ng/mL for E. coli O157:H7 and Stx1, respectively, in a ~75 min total assay time.http://www.mdpi.com/1424-8220/15/12/29807antibody microarraybacteriacentrifugationfluorescenceimmunoassaymultiwellmicrotiter platemultiplextoxin |
spellingShingle | Andrew G. Gehring Jeffrey D. Brewster Yiping He Peter L. Irwin George C. Paoli Tawana Simons Shu-I Tu Joseph Uknalis Antibody Microarray for E. coli O157:H7 and Shiga Toxin in Microtiter Plates Sensors antibody microarray bacteria centrifugation fluorescence immunoassay multiwell microtiter plate multiplex toxin |
title | Antibody Microarray for E. coli O157:H7 and Shiga Toxin in Microtiter Plates |
title_full | Antibody Microarray for E. coli O157:H7 and Shiga Toxin in Microtiter Plates |
title_fullStr | Antibody Microarray for E. coli O157:H7 and Shiga Toxin in Microtiter Plates |
title_full_unstemmed | Antibody Microarray for E. coli O157:H7 and Shiga Toxin in Microtiter Plates |
title_short | Antibody Microarray for E. coli O157:H7 and Shiga Toxin in Microtiter Plates |
title_sort | antibody microarray for e coli o157 h7 and shiga toxin in microtiter plates |
topic | antibody microarray bacteria centrifugation fluorescence immunoassay multiwell microtiter plate multiplex toxin |
url | http://www.mdpi.com/1424-8220/15/12/29807 |
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