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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.