Quantification of Vibrio cholerae cholix exotoxin by sandwich bead-ELISA
Quantification of Vibrio cholerae cholix exotoxin by sandwich bead-ELISA
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夹心珠-ELISA 定量霍乱弧菌胆汁外毒素
DOI：
10.1099/jmm.0.001311
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发表时间：
2021
影响因子：
3
通讯作者：
Yamasaki Shinji
中科院分区：
文献类型：
--
作者：
Awasthi Sharda Prasad;Chowdhury Nityananda;Hatanaka Noritoshi;Hinenoya Atsushi;Ramamurthy Thandavarayan;Asakura Masahiro;Yamasaki Shinji
介绍。胆毒素（ChxA）是一种由霍乱弧菌产生的adp核糖基化外毒素。然而，到目前为止，还没有可用于ChxA的定量分析，这使得很难检测和估计由v产生的ChxA的水平。霍乱弧菌。假设/差距语句。建立一种可靠、特异的检测ChxA产生水平的方法，有助于了解ChxA在霍乱发病机制中的作用。目的：建立一种基于头部的夹心ELISA （head -ELISA）定量检测ChxA，并评价ChxA在霍乱发病机制中的重要性。choleraeinfection.Methodology。用新西兰大白兔培养抗rchxa抗体，用马来酰亚胺法制备fab -马萝卜过氧化物酶偶联物，用于珠状酶联免疫吸附测定。本抗-ChxA珠状酶联免疫吸附测定法用于测定各种病毒产生的ChxA。choleraestrains。在碱性蛋白胨水（APW）、Luria-Bertani肉汤和AKI培养基中测定了ChxA的产量。最后，采用具有代表性的小鼠致死性实验对该方法进行评价。基于抗chxa抗体珠状酶联免疫吸附试验，将霍乱菌株分为低至高chxa产生菌。建立了一种灵敏的珠状酶联免疫吸附试验，可以定量0.6 ~ 60 ng ml−1的ChxA。ChxA主要在细胞外无细胞上清中检测到，其产生水平从1.2 ng ml - 1到1.6µg ml - 1不等。ChxA产量最高时，v。霍乱菌在LB肉汤中培养，但在APW或AKI培养基中没有培养。ChxA-producerV。在小鼠模型实验中，霍乱菌株的致死率为20 - 80%，只有高ChxA ii产生物比非ChxA-产生物具有统计学上的致死率。结论：ChxA I和ChxA II的产生量主要在细胞外产生，其产生量随菌株和培养条件的不同而不同。本研究建立的珠状酶联免疫吸附试验可用于ChxA v型霍乱菌的检测和定量。
Introduction.Cholix toxin (ChxA) is an ADP-ribosylating exotoxin produced byVibrio cholerae. However, to date, there is no quantitative assay available for ChxA, which makes it difficult to detect and estimate the level of ChxA produced byV. cholerae.Hypothesis/Gap Statement.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 inV. choleraepathogenesis.Aim.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 ofV. choleraeinfection.Methodology.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 variousV. choleraestrains. 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 representativeV. choleraestrains categorized as low to high ChxA-producers based on anti-ChxA bead-ELISA.Results.A sensitive bead-ELISA assay, which can quantify from 0.6 to 60 ng ml−1of ChxA, was developed. ChxA was mostly detected in the extracellular cell-free supernatant and its production level varied from 1.2 ng ml−1to 1.6 µg ml−1. The highest ChxA production was observed whenV. choleraestrains were cultured in LB broth, but not in APW or AKI medium. The ChxA-producerV. choleraestrains 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.Conclusion.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 inV. choleraestrains.