title: A high-throughput, precipitating colorimetric sandwich ELISA microarray for Shiga toxins.
abstract: Shiga toxins 1 and 2 (Stx1 and Stx2) from Shiga toxin-producing E. coli (STEC) bacteria were simultaneously detected with a newly developed, high-throughput antibody microarray platform. The proteinaceous toxins were immobilized and sandwiched between biorecognition elements (monoclonal antibodies) and pooled horseradish peroxidase (HRP)-conjugated monoclonal antibodies. Following the reaction of HRP with the precipitating chromogenic substrate (metal enhanced 3,3-diaminobenzidine tetrahydrochloride or DAB), the formation of a colored product was quantitatively measured with an inexpensive flatbed page scanner. The colorimetric ELISA microarray was demonstrated to detect Stx1 and Stx2 at levels as low as ~4.5 ng/mL within ~2 h of total assay time with a narrow linear dynamic range of ~1-2 orders of magnitude and saturation levels well above background. Stx1 and/or Stx2 produced by various strains of STEC were also detected following the treatment of cultured cells with mitomycin C (a toxin-inducing antibiotic) and/or B-PER (a cell-disrupting, protein extraction reagent). Semi-quantitative detection of Shiga toxins was demonstrated to be sporadic among various STEC strains following incubation with mitomycin C; however, further reaction with B-PER generally resulted in the detection of or increased detection of Stx1, relative to Stx2, produced by STECs inoculated into either axenic broth culture or culture broth containing ground beef.
authors.affiliation: 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. andrew.gehring@ars.usda.gov.
authors.forename: Andrew
authors.initials: A
authors.lastname: Gehring
authors.affiliation: Foodborne Toxin Detection and Prevention Research Unit, United States Department of Agriculture-Pacific West Area-Agricultural Research Service-Western Regional Research Center, Albany, CA 94710, USA. xiaohua.he@ars.usda.gov.
authors.forename: Xiaohua
authors.initials: X
authors.lastname: He
authors.affiliation: 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. pina.fratamico@ars.usda.gov.
authors.forename: Pina
authors.initials: P
authors.lastname: Fratamico
authors.affiliation: 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. joe.lee@ars.usda.gov.
authors.forename: Joseph
authors.initials: J
authors.lastname: Lee
authors.affiliation: 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. lori.bagi@ars.usda.gov.
authors.forename: Lori
authors.initials: L
authors.lastname: Bagi
authors.affiliation: 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. jeffrey.brewster@ars.usda.gov.
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Brewster
authors.affiliation: 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. george.paoli@ars.usda.gov.
authors.forename: George
authors.initials: G
authors.lastname: Paoli
authors.affiliation: 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. 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-North Atlantic Area-Agricultural Research Service-Eastern Regional Research Center, Wyndmoor, PA 19038, USA. yanping.xie@ars.usda.gov.
authors.forename: Yanping
authors.initials: Y
authors.lastname: Xie
authors.affiliation: Foodborne Toxin Detection and Prevention Research Unit, United States Department of Agriculture-Pacific West Area-Agricultural Research Service-Western Regional Research Center, Albany, CA 94710, USA. craig.skinner@ars.usda.gov.
authors.forename: Craig
authors.initials: C
authors.lastname: Skinner
authors.affiliation: NanoDetection Technology, Inc., Franklin, OH 45005, USA. cbarnett1951@gmail.com.
authors.forename: Charlie
authors.initials: C
authors.lastname: Barnett
authors.affiliation: NanoDetection Technology, Inc., Franklin, OH 45005, USA. dough@ndtbio.com.
authors.forename: Douglas
authors.initials: D
authors.lastname: Harris
mesh_terms: D015100:3,3'-Diaminobenzidine / Q000737:chemistry; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D000918:Antibody Specificity; D003124:Colorimetry; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D005513:Food Inspection / Q000379:methods*; D057166:High-Throughput Screening Assays; D047468:Immunoprecipitation; D007202:Indicators and Reagents / Q000737:chemistry; D057230:Limit of Detection; D016685:Mitomycin / Q000494:pharmacology; D040081:Protein Array Analysis; D015203:Reproducibility of Results; D022622:Shiga Toxin 1 / Q000819:agonists / Q000032:analysis* / Q000378:metabolism; D022641:Shiga Toxin 2 / Q000819:agonists / Q000032:analysis* / Q000378:metabolism; D054323:Shiga-Toxigenic Escherichia coli / Q000187:drug effects / Q000378:metabolism; D014481:United States; D016885:United States Department of Agriculture
chemical_list: D000900:Anti-Bacterial Agents; D000911:Antibodies, Monoclonal; D007202:Indicators and Reagents; D022622:Shiga Toxin 1; D022641:Shiga Toxin 2; D015100:3,3'-Diaminobenzidine; D016685:Mitomycin