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A High-Throughput, Precipitating Colorimetric Sandwich ELISA Microarray for Shiga Toxins

Andrew Gehring, Xiaohua He, Pina Fratamico, Joseph Lee, Lori Bagi, Jeffery Brewster, Yiping He, Yanping Xie, Craig Skinner, Charlie Barnett, Douglas Harris
Toxins 2014 v.6 no.6 pp. 1855-1872
peroxidase, antibody microarrays, colorimetry, antibiotics, ground beef, enzyme-linked immunosorbent assay, monoclonal antibodies, cultured cells, mitomycin, verotoxins, culture media, bacteria, Shiga toxin-producing Escherichia coli
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.