{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,5,14]],"date-time":"2025-05-14T00:08:45Z","timestamp":1747181325629,"version":"3.40.5"},"reference-count":18,"publisher":"Microbiology Society","issue":"4","funder":[{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"crossref","award":["26460533"],"award-info":[{"award-number":["26460533"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2021,4,8]]},"abstract":"\n Introduction.<\/jats:bold> Cholix toxin (ChxA) is an ADP-ribosylating exotoxin produced by \n \n Vibrio cholerae<\/jats:ext-link>\n <\/jats:named-content>\n <\/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 \n \n V. cholerae<\/jats:ext-link>\n <\/jats:named-content>\n <\/jats:italic>.<\/jats:p>\n \n 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 \n \n V. cholerae<\/jats:ext-link>\n <\/jats:named-content>\n <\/jats:italic> pathogenesis.<\/jats:p>\n \n 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 \n \n V. cholerae<\/jats:ext-link>\n <\/jats:named-content>\n <\/jats:italic> infection.<\/jats:p>\n \n 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 \n \n V. cholerae<\/jats:ext-link>\n <\/jats:named-content>\n <\/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 \n \n V. cholerae<\/jats:ext-link>\n <\/jats:named-content>\n <\/jats:italic> strains categorized as low to high ChxA-producers based on anti-ChxA bead-ELISA.<\/jats:p>\n \n Results.<\/jats:bold> A sensitive bead-ELISA assay, which can quantify from 0.6 to 60 ng ml\u22121<\/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\u22121<\/jats:sup> to 1.6 \u00b5g ml\u22121<\/jats:sup>. The highest ChxA production was observed when \n \n V. cholerae<\/jats:ext-link>\n <\/jats:named-content>\n <\/jats:italic> strains were cultured in LB broth, but not in APW or AKI medium. The ChxA-producer \n \n V. cholerae<\/jats:ext-link>\n <\/jats:named-content>\n <\/jats:italic> strains showed 20\u201380\u200a%\u2009lethality 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.<\/jats:p>\n \n Conclusion.<\/jats:bold> 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 \n \n V. cholerae<\/jats:ext-link>\n <\/jats:named-content>\n <\/jats:italic> strains.<\/jats:p>","DOI":"10.1099\/jmm.0.001311","type":"journal-article","created":{"date-parts":[[2021,4,8]],"date-time":"2021-04-08T17:18:13Z","timestamp":1617902293000},"source":"Crossref","is-referenced-by-count":0,"title":["Quantification of Vibrio cholerae cholix exotoxin by sandwich bead-ELISA"],"prefix":"10.1099","volume":"70","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-1606-3543","authenticated-orcid":false,"given":"Sharda Prasad","family":"Awasthi","sequence":"first","affiliation":[{"name":"Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nityananda","family":"Chowdhury","sequence":"additional","affiliation":[{"name":"Present address: Medical University of South Carolina, 173 Ashley Ave., BSB 246, Charleston, SC, USA"},{"name":"Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4708-8487","authenticated-orcid":false,"given":"Noritoshi","family":"Hatanaka","sequence":"additional","affiliation":[{"name":"Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1149-984X","authenticated-orcid":false,"given":"Atsushi","family":"Hinenoya","sequence":"additional","affiliation":[{"name":"Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Thandavarayan","family":"Ramamurthy","sequence":"additional","affiliation":[{"name":"National Institute of Cholera and Enteric Diseases, Kolkata, India"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Masahiro","family":"Asakura","sequence":"additional","affiliation":[{"name":"Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9063-2201","authenticated-orcid":false,"given":"Shinji","family":"Yamasaki","sequence":"additional","affiliation":[{"name":"Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"345","reference":[{"key":"R1","doi-asserted-by":"publisher","DOI":"10.11150\/kansenshogakuzasshi1970.71.1037"},{"key":"R2","article-title":"(February 2014) Media centre, fact sheets, N\u00b0107 (Cholera). 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