A method for detecting Shiga toxin and Shiga-like toxin-I in pure and mixed culture.
Shiga toxin and Shiga-like toxins (SLTs, syn. Verotoxins) are currently detected by tissue culture assays that are expensive, time-consuming and require specialised facilities and experienced personnel. We have developed a rapid method to detect Shiga toxin and SLT-I (Verotoxin 1) based on their binding to globotriosyl ceramide (Gb3). Bound toxin was then detected by an enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies. The direct detection of cytotoxins from pure culture plates and from a mixed bacterial culture was studied. Using polymyxin extraction (0.1 g/L, 30 min, 37 degrees C) and Gb3-based ELISA we detected toxin from reference strains Shigella dysenteriae 1 strain 60R (Shiga toxin) and Escherichia coli O26:H11 strain H30 (SLT-I), and from clinical isolates of E. coli O157:H7 and O26:H11 (both SLT-I) from 11 patients with diarrhoea, haemorrhagic colitis or haemolytic uraemic syndrome. Toxin production by these strains was confirmed by a radiolabelled HeLa cell assay and the structural genes were detected by DNA hybridisation. The Gb3-based ELISA could detect SLT-I in extracts of a mixed culture even when the toxin-positive strains represented only 1% of the mixture. No cross-reactivity was found with bacteria that produce other cytotoxins, such as other E. coli and Shigella, Salmonella, Aeromonas and Campylobacter spp.
Ashkenazi
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Department of Pediatrics, University of Texas Medical School, Houston 77030.
Cleary
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D001427:Bacterial Toxins / Q000032:analysis* / Q000235:genetics; D003603:Cytotoxins / Q000032:analysis*; D004768:Enterotoxins / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D009693:Nucleic Acid Hybridization; D011112:Polymyxin B / Q000494:pharmacology; D022622:Shiga Toxin 1; D022603:Shiga Toxins
D001427:Bacterial Toxins; D003603:Cytotoxins; D004768:Enterotoxins; D022622:Shiga Toxin 1; D022603:Shiga Toxins; D011112:Polymyxin B