Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan.
Present address: Medical University of South Carolina, 173 Ashley Ave., BSB 246, Charleston, SC, USA.
J Med Microbiol. 2021 Apr;70(4). doi: 10.1099/jmm.0.001311.
Cholix toxin (ChxA) is an ADP-ribosylating exotoxin produced by . However, to date, there is no quantitative assay available for ChxA, which makes it difficult to detect and estimate the level of ChxA produced by . It is important to develop a reliable and specific quantitative assay to measure the production level of ChxA, which will help us to understand the role of ChxA in pathogenesis. The aim of this study was to develop a bead-based sandwich ELISA (bead-ELISA) for the quantification of ChxA and to evaluate the importance of ChxA in the pathogenesis of infection. Anti-rChxA was raised in New Zealand white rabbits, and Fab-horse radish peroxidase conjugate was prepared by the maleimide method to use in the bead-ELISA. This anti-ChxA bead-ELISA was applied to quantify the ChxA produced by various strains. The production of ChxA was examined in different growth media such as alkaline peptone water (APW), Luria-Bertani broth and AKI. Finally, the assay was evaluated using a mouse lethality assay with representative strains categorized as low to high ChxA-producers based on anti-ChxA bead-ELISA. A sensitive bead-ELISA assay, which can quantify from 0.6 to 60 ng ml of ChxA, was developed. ChxA was mostly detected in the extracellular cell-free supernatant and its production level varied from 1.2 ng ml to 1.6 µg ml. The highest ChxA production was observed when strains were cultured in LB broth, but not in APW or AKI medium. The ChxA-producer strains showed 20-80 % lethality and only the high ChxA II-producer was statistically more lethal than a non-ChxA-producer, in the mice model assay. ChxA I and II production levels were not well correlated with mice lethality, and this could be due to the heterogeneity of the strains tested. ChxA I to III was produced mostly extracellularly at various levels depending on strains and culture conditions. The bead-ELISA developed in this study is useful for the detection and quantification of ChxA in strains.
胆盐水解酶毒素（ChxA）是一种由 产生的 ADP-核糖基化外毒素。然而，迄今为止，还没有可用于检测 ChxA 的定量分析方法，这使得难以检测和估计 产生的 ChxA 水平。开发一种可靠和特异的定量分析方法来测量 ChxA 的产生水平非常重要，这将有助于我们了解 ChxA 在 发病机制中的作用。本研究旨在开发一种基于珠子的夹心 ELISA（珠子 ELISA）来定量检测 ChxA，并评估 ChxA 在 感染发病机制中的重要性。用新西兰白兔制备了抗 rChxA，并用马来酰亚胺法制备了 Fab-辣根过氧化物酶缀合物，用于珠子 ELISA。该抗 ChxA 珠子 ELISA 用于定量检测各种 菌株产生的 ChxA。在碱性蛋白胨水 (APW)、Luria-Bertani 肉汤和 AKI 等不同生长培养基中检查 ChxA 的产生。最后，使用具有代表性的 菌株的小鼠致死性测定来评估该测定，这些菌株根据抗 ChxA 珠子 ELISA 分为低产 ChxA 菌株和高产 ChxA 菌株。开发了一种灵敏的珠子 ELISA 测定法，可定量检测 0.6 至 60ng/ml 的 ChxA。ChxA 主要在细胞外无细胞上清液中检测到，其产生水平从 1.2ng/ml 到 1.6μg/ml 不等。当 菌株在 LB 肉汤中培养时，观察到最高的 ChxA 产生，但在 APW 或 AKI 培养基中则没有。在小鼠模型测定中，ChxA 产生菌表现出 20-80%的致死率，只有高产 ChxA II 的菌株比非 ChxA 产生菌具有统计学上更高的致死率。ChxA I 和 II 的产生水平与小鼠致死率没有很好的相关性，这可能是由于测试菌株的异质性所致。ChxA I 至 III 主要在不同水平上以细胞外的形式产生，具体取决于菌株和培养条件。本研究中开发的珠子 ELISA 可用于检测和定量检测 菌株中的 ChxA。
