Rapid method to detect shiga toxin and shiga-like toxin I based on binding to globotriosyl ceramide (Gb3) their natural receptor.

摘要

Shiga toxin and the closely related Shiga-like toxins produced by Escherichia coli represent a group of very similar cytotoxins that may play an important role in diarrheal disease and hemolytic uremic syndrome. These toxins have the same biologic activities and according to recent studies also share the same binding receptor, globotriosyl ceramide (Gb3). They are currently detected, on the basis of their ability to damage several cell lines, by using expensive and tedious assays that require facilities for and experience with tissue cultures and are therefore most suitable for research laboratories. We have developed a rapid method to detect Shiga toxin and Shiga-like toxin I based on specific binding to their Gb3 natural receptor, which was coated onto microdilution plates. Bound toxin was then detected by enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies. The sensitivity of the Gb3 ELISA was 0.2 ng (2 ng/ml) of purified toxin. The assay was positive with sonic extracts of Shigella dysenteriae serotype 1 strain 6OR (a Shiga toxin producer), E. coli serotype O26:H11 strain H30, and E. coli serotype O157:H7 (both Shiga-like toxin I producers). The assay was very specific in that no cross-reactivity was noted with purified cholera toxin, E. coli heat-labile and heat-stable enterotoxins, and Clostridium difficile cytotoxin, or sonic extracts of other cytotoxin-producing organisms, such as other shigellae, pathogenic and nonpathogenic E. coli, Salmonella spp., Campylobacter spp., and Aeromonas spp. These results were in complete agreement with a [3H]thymidine-labeled HeLa cell cytotoxicity assay and with detection of the structural genes by DNA hybridization studies with a Shiga-like toxin I probe. Quantitative analysis showed a high correlation between Gb3 ELISA and HeLa cell assay when fractions obtained at various stages of toxin purification were examined by both methods (r = 0.99, P < 0.01). This rapid Gb3 ELISA is sensitive and specific and may be diagnostically useful in cytotoxin-related infections.