Title: A high-throughput, precipitating colorimetric sandwich ELISA microarray for shiga toxins Authors
|Barnett, Charlie -|
|Harris, Douglas -|
Submitted to: Toxins
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 2, 2014
Publication Date: June 11, 2014
Citation: Gehring, A.G., He, X., Fratamico, P.M., Lee, J., Bagi, L.K., Brewster, J.D., Paoli, G., He, Y., Xie, Y., Skinner, C.B., Barnett, C., Harris, D. 2014. A high-throughput, precipitating colorimetric sandwich ELISA microarray for shiga toxins. Toxins. DOI: 10.3390/toxins6061855. Interpretive Summary: Ingestion of foods containing the harmful bacteria Shiga toxin-producing E. coli (STEC), is a serious threat to human health. STEC may, in part, be identified by the various Shiga toxins (Stx) that they produce. A new technique, known as “colorimetric ELISA microarray” (CEM), was developed and it can be used to screen foods for these toxins. CEM, a rapid and relatively inexpensive technique, was demonstrated to directly detect very low amounts of Stx within ~1-2 h of total testing time. The technique was also used to rapidly detect Stx produced by different STEC that were grown in a culture media with or without ground beef. Armed with CEM, regulators may screen very large numbers of food samples for Shiga toxins in a few hours.
Technical 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 reaction of HRP with precipitating chromogenic substrate (metal enhanced 3,3-diaminobenzidine tetrahydrochloride or DAB), formation of 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 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 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.