Shiga toxin-producing Escherichia coli (STEC) is a major intestinal pathogen and causes serious gastrointestinal illness, which includes diarrhea, hemorrhagic colitis, and life-threatening hemolytic uremic syndrome. The major virulence factors of STEC are Shiga toxins (Stx1 and Stx2), which belong to the AB-type toxin family. Among several subtypes of Stx1 and Stx2, the production of Stx2a is thought to be a risk factor for severe STEC infections, but Stx2a production levels vary markedly between STEC strains, even strains with the same serotype. Therefore, quantitative analyses of Stx2 production by STEC strains are important to understand the virulence potential of specific lineages or sublineages. In this study, we developed a novel Stx2 quantification method by utilizing homogeneous time-resolved fluorescence resonance energy transfer (HTRF) technology. To determine suitable “sandwich” assay conditions, we tested 6 combinations of fluorescence-labeled monoclonal antibodies (mAbs) specific to Stx2 and compared the HTRF signal intensities obtained at various incubation times. Through this analysis, we selected the most suitable mAb pair, one recognizing the A subunit and the other recognizing the B subunit, thus together detecting Stx holotoxins. The optimal incubation time was also determined (18 h). Then, we optimized the concentrations of the two mAbs based on the range for linearity. The established HTRF assay detected 0.5 ng/ml of the highly purified recombinant Stx2a and Stx2e proteins and the working range was 1–64 ng/ml for both Stx2a and Stx2e. Through the quantification analysis of Stx proteins in STEC cell lysates, we confirmed that other Stx2 subtypes (Stx2b, Stx2c, Stx2d and Stx2g) can also be quantified at a certain level of accuracy, while this assay system does not detect Stx2f, which is highly divergent in sequence from other Stx2 subtypes, and Stx1. As the HTRF protocol we established is simple, this assay system should prove useful for the quantitative analysis of Stx2 production levels of a large number of STEC strains.

中文翻译：

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开发用于定量大肠杆菌产生的志贺毒素 2 的均相时间分辨 FRET (HTRF) 检测

产志贺毒素大肠杆菌 (STEC) 是一种主要的肠道病原体，会导致严重的胃肠道疾病，包括腹泻、出血性结肠炎和危及生命的溶血性​​尿毒症综合征。STEC 的主要毒力因子是志贺毒素（Stx1 和 Stx2），属于 AB 型毒素家族。在 Stx1 和 Stx2 的几个亚型中，Stx2a 的产生被认为是严重 STEC 感染的危险因素，但 STEC 菌株之间的 Stx2a 产生水平显着不同，即使是具有相同血清型的菌株。因此，对 STEC 菌株产生的 Stx2 进行定量分析对于了解特定谱系或亚谱系的毒力潜力很重要。在这项研究中，我们利用均相时间分辨荧光共振能量转移 (HTRF) 技术开发了一种新的 Stx2 量化方法。为了确定合适的“夹心”检测条件，我们测试了 6 种对 Stx2 具有特异性的荧光标记单克隆抗体 (mAb) 组合，并比较了在不同孵育时间获得的 HTRF 信号强度。通过这种分析，我们选择了最合适的 mAb 对，一个识别 A 亚基，另一个识别 B 亚基，从而一起检测 Stx 全毒素。还确定了最佳孵育时间（18 小时）。然后，我们根据线性范围优化了两种 mAb 的浓度。已建立的 HTRF 检测检测到 0.5 ng/ml 的高度纯化的重组 Stx2a 和 Stx2e 蛋白，Stx2a 和 Stx2e 的工作范围为 1–64 ng/ml。通过对 STEC 细胞裂解物中 Stx 蛋白的定量分析，我们确认其他 Stx2 亚型（Stx2b、Stx2c、Stx2d 和 Stx2g）也可以在一定的准确度下进行定量，而该检测系统不检测 Stx2f，这是高度在序列上与其他 Stx2 亚型和 Stx1 不同。由于我们建立的 HTRF 协议很简单，因此该检测系统应该可用于对大量 STEC 菌株的 Stx2 生产水平进行定量分析。