Ashkenazi S, Cleary T G
Department of Pediatrics, University of Texas Medical School, Houston 77030.
J Clin Microbiol. 1989 Jun;27(6):1145-50. doi: 10.1128/jcm.27.6.1145-1150.1989.
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.
志贺毒素以及大肠杆菌产生的与之密切相关的志贺样毒素代表了一组非常相似的细胞毒素，它们可能在腹泻病和溶血尿毒综合征中起重要作用。这些毒素具有相同的生物学活性，并且根据最近的研究，它们还共享相同的结合受体，即球三糖神经酰胺（Gb3）。目前，基于它们对几种细胞系的损伤能力，通过使用昂贵且繁琐的检测方法来检测它们，这些方法需要组织培养设施和经验，因此最适合研究实验室。我们开发了一种基于与包被在微量稀释板上的Gb3天然受体特异性结合来检测志贺毒素和志贺样毒素I的快速方法。然后用单克隆抗体通过酶联免疫吸附测定（ELISA）检测结合的毒素。Gb3 ELISA的灵敏度为0.2 ng（2 ng/ml）纯化毒素。该检测方法对痢疾志贺菌1型菌株6OR（一种志贺毒素产生菌）、大肠杆菌O26:H11菌株H30和大肠杆菌O157:H7（均为志贺样毒素I产生菌）的超声提取物呈阳性。该检测方法非常特异，与纯化的霍乱毒素、大肠杆菌不耐热和耐热肠毒素、艰难梭菌细胞毒素或其他产生细胞毒素的生物体（如其他志贺菌、致病性和非致病性大肠杆菌、沙门氏菌属、弯曲杆菌属和气单胞菌属）的超声提取物均无交叉反应。这些结果与[3H]胸苷标记的HeLa细胞细胞毒性检测以及用志贺样毒素I探针进行DNA杂交研究检测结构基因完全一致。定量分析表明，当用两种方法检测毒素纯化不同阶段获得的组分时，Gb3 ELISA与HeLa细胞检测之间具有高度相关性（r = 0.99，P < 0.01）。这种快速的Gb3 ELISA灵敏且特异，可能在细胞毒素相关感染的诊断中有用。
