title: Antibody Microarray for E. coli O157:H7 and Shiga Toxin in Microtiter Plates.
abstract: 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.
authors.affiliation: Molecular Characterization of Foodborne Pathogens Research Unit, United States Department of Agriculture-Northeast Area, Agricultural Research Service, Eastern Regional Research Center, Wyndmoor, PA 19038, USA. andrew.gehring@ars.usda.gov.
authors.forename: Andrew G
authors.initials: AG
authors.lastname: Gehring
authors.affiliation: Molecular Characterization of Foodborne Pathogens Research Unit, United States Department of Agriculture-Northeast Area, Agricultural Research Service, Eastern Regional Research Center, Wyndmoor, PA 19038, USA. jeffrey.brewster@ars.usda.gov.
authors.forename: Jeffrey D
authors.initials: JD
authors.lastname: Brewster
authors.affiliation: Molecular Characterization of Foodborne Pathogens Research Unit, United States Department of Agriculture-Northeast Area, Agricultural Research Service, Eastern Regional Research Center, Wyndmoor, PA 19038, USA. yiping.he@ars.usda.gov.
authors.forename: Yiping
authors.initials: Y
authors.lastname: He
authors.affiliation: Molecular Characterization of Foodborne Pathogens Research Unit, United States Department of Agriculture-Northeast Area, Agricultural Research Service, Eastern Regional Research Center, Wyndmoor, PA 19038, USA. peter.irwin@ars.usda.gov.
authors.forename: Peter L
authors.initials: PL
authors.lastname: Irwin
authors.affiliation: Molecular Characterization of Foodborne Pathogens Research Unit, United States Department of Agriculture-Northeast Area, Agricultural Research Service, Eastern Regional Research Center, Wyndmoor, PA 19038, USA. george.paoli@ars.usda.gov.
authors.forename: George C
authors.initials: GC
authors.lastname: Paoli
authors.affiliation: Molecular Characterization of Foodborne Pathogens Research Unit, United States Department of Agriculture-Northeast Area, Agricultural Research Service, Eastern Regional Research Center, Wyndmoor, PA 19038, USA. tawana.simons@ars.usda.gov.
authors.forename: Tawana
authors.initials: T
authors.lastname: Simons
authors.affiliation: Molecular Characterization of Foodborne Pathogens Research Unit, United States Department of Agriculture-Northeast Area, Agricultural Research Service, Eastern Regional Research Center, Wyndmoor, PA 19038, USA. shui.tu@ars.usda.gov.
authors.forename: Shu-I
authors.initials: SI
authors.lastname: Tu
authors.affiliation: Molecular Characterization of Foodborne Pathogens Research Unit, United States Department of Agriculture-Northeast Area, Agricultural Research Service, Eastern Regional Research Center, Wyndmoor, PA 19038, USA. joseph.uknalis@ars.usda.gov.
authors.forename: Joseph
authors.initials: J
authors.lastname: Uknalis
mesh_terms: D002498:Centrifugation; D019453:Escherichia coli O157 / Q000276:immunology / Q000302:isolation & purification*; D057166:High-Throughput Screening Assays; D007118:Immunoassay / Q000379:methods*; D046228:Microarray Analysis / Q000379:methods*; D022621:Shiga Toxin / Q000032:analysis* / Q000276:immunology
chemical_list: D022621:Shiga Toxin
keywords: antibody microarray; bacteria; centrifugation; fluorescence; immunoassay; microtiter plate; multiplex; multiwell; toxin