Quantification of Vibrio cholerae cholix exotoxin by sandwich bead-ELISA

<jats:p> <jats:bold>Introduction.</jats:bold> Cholix toxin (ChxA) is an ADP-ribosylating exotoxin produced by <jats:italic> <jats:named-content content-type="species"> <jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://doi.org/10.1601/nm.2947" xlink:type="simple">Vibrio cholerae</jats:ext-link> </jats:named-content> </jats:italic>. 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 <jats:italic> <jats:named-content content-type="species"> <jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://doi.org/10.1601/nm.2947" xlink:type="simple">V. cholerae</jats:ext-link> </jats:named-content> </jats:italic>.</jats:p> <jats:p> <jats:bold>Hypothesis/Gap Statement.</jats:bold> 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 <jats:italic> <jats:named-content content-type="species"> <jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://doi.org/10.1601/nm.2947" xlink:type="simple">V. cholerae</jats:ext-link> </jats:named-content> </jats:italic> pathogenesis.</jats:p> <jats:p> <jats:bold>Aim.</jats:bold> 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 <jats:italic> <jats:named-content content-type="species"> <jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://doi.org/10.1601/nm.2947" xlink:type="simple">V. cholerae</jats:ext-link> </jats:named-content> </jats:italic> infection.</jats:p> <jats:p> <jats:bold>Methodology.</jats:bold> 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 <jats:italic> <jats:named-content content-type="species"> <jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://doi.org/10.1601/nm.2947" xlink:type="simple">V. cholerae</jats:ext-link> </jats:named-content> </jats:italic> 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 <jats:italic> <jats:named-content content-type="species"> <jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://doi.org/10.1601/nm.2947" xlink:type="simple">V. cholerae</jats:ext-link> </jats:named-content> </jats:italic> strains categorized as low to high ChxA-producers based on anti-ChxA bead-ELISA.</jats:p> <jats:p> <jats:bold>Results.</jats:bold> A sensitive bead-ELISA assay, which can quantify from 0.6 to 60 ng ml<jats:sup>−1</jats:sup> of ChxA, was developed. ChxA was mostly detected in the extracellular cell-free supernatant and its production level varied from 1.2 ng ml<jats:sup>−1</jats:sup> to 1.6 µg ml<jats:sup>−1</jats:sup>. The highest ChxA production was observed when <jats:italic> <jats:named-content content-type="species"> <jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://doi.org/10.1601/nm.2947" xlink:type="simple">V. cholerae</jats:ext-link> </jats:named-content> </jats:italic> strains were cultured in LB broth, but not in APW or AKI medium. The ChxA-producer <jats:italic> <jats:named-content content-type="species"> <jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://doi.org/10.1601/nm.2947" xlink:type="simple">V. cholerae</jats:ext-link> </jats:named-content> </jats:italic> 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 het ...