A Rapid Bioluminescent Enzyme Immunoassay (BLEIA) for the Detection of Shiga Toxin Types 1 and 2

Abstract

In recent years, Escherichia coli 0157: 87 has emerged as a global public health concern. Among the more important virulence characteristics of this strain is its ability to produce one or more Shiga toxins (Stx). Traditional culture-based methods for assay of enteric toxins in foods and clinical samples are relatively slow and results can be ambiguous. In this work, we established a toxin-detection system based on bioluminescent enzyme immunoassay (BLEIA) using a simple and inexpensive device. The system could detect both Shiga toxin types 1 and 2 individually within 150 min with a detection limit for each toxin at 5 pglml. In our study of previously characterized Shigatoxigenic and all non-Shigatoxigenic E. coli and other bacterial species, we found all Shigatoxigenic strains to be positive and non-Shigatoxigenic E. coli and other bacterial species to be negative. This assay was also used to detect Stxs in milk and supernatant fluids from minced chicken and beef. For clinical stool samples we noted a tendency for the system to give unexpectedly high background level. Our results suggest the feasibility of using BLEIA methodology for the simple, rapid and sensitive detection of toxins from culture supernatant, various foods and clinical samples.

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

“…Array analysis grids were manually positioned for data signal/noise analysis, a process that is more challenging at response levels for the lowest concentrations tested, since they were somewhat faint. Monoclonal antibodies (mAbs) were employed in this example, and as expected, immunoassays generated by other groups using polyclonal antibodies and/or more sensitive, though more expensive, luminescence detection fared better, with reported detection limits at the pg/mL level [26,27,28]. …”

EVALUATION OF COLONOSCOPIC FINDINGS IN PATIENTS WITH DIARRHEAGENIC ESCHERICHIA COLI‐INDUCED HEMORRHAGIC COLITIS

“…That is, in tests detecting the LPS antigen of O157, low bacterial quantity in the stool specimen can result in a false negative, 20 or a cross-reaction to Salmonella or Citrobacter can lead to false positive results. 19,20 Tests for detecting Stx usually require that a bacterial colony forms on the culture medium, 21,22 thus taking more time to make a diagnosis of EHEC infection. Some attempts have been made to apply these tests directly to the detection of Stx in a patient's stool infected by EHEC, but it has been pointed out that the tests' sensitivity for detecting Stx is usually low or ambiguous.…”

“…Some attempts have been made to apply these tests directly to the detection of Stx in a patient's stool infected by EHEC, but it has been pointed out that the tests' sensitivity for detecting Stx is usually low or ambiguous. 22,23 Used to augment the rapid laboratory tests described above, characteristic colonoscopic findings of EHEC-induced hemorrhagic colitis could provide physicians with more tools allowing for earlier detection of EHEC infection. The information gathered by these specific findings during colonoscopy in patients with bloody diarrhea could provide validating data and could demonstrate that EHEC is probably the causative pathogen.…”

Rapid detection of virulence stx2 gene of Enterohemorrhagic Escherichia coli using two-step ultra-rapid real-time PCR

“…To facilitate the detection of EHEC, enzyme immunoassays (EIA) (Yamazaki et al 2001), enzyme-linked immunosorbent assay (ELISA) (Perera et al 1988), and real-time PCR diagnostic methods (Bellin et al 2001) are usually used instead of the conventional sorbitol/MacConkey agar culture (SMAC) screening (James et al 1998;March and Ratnam 1986). EIA and ELISA methods have advantages of simplicity and speed but they are less sensitive than SMAC screening (Klein et al 2004).…”