Evidence for the presence of a receptor for the cytolethal distending toxin (CLDT) of Campylobacter jejuni on CHO and HeLa cell membranes and development of a receptor-based enzyme-linked immunosorbent assay for detection of CLDT.
Using ligand blotting, it was found that partially purified cytolethal distending toxin prepared from an enterotoxigenic strain of Campylobacter jejuni, bound to two peptides of molecular masses of approximately 59 kDa and 45 kDa and to a single peptide of 59 kDa in protein blots prepared from HeLa and CHO cell membranes, respectively. In contrast, labile toxin of Escherichia coli and cholera toxin bound to a single peptide of the same molecular mass (15 kDa) on protein blots prepared from both CHO and HeLa cell crude membranes resolved by gel electrophoresis. This banding pattern was identical using SDS-solubilized membrane, with or without heat treatment, but no band was obtained when reduced (treatment with 2-mercaptoethanol) samples were used for the gel electrophoresis. The differences between receptors of cytolethal distending toxin and cholera toxin/labile toxin were exploited to develop a receptor-based enzyme-linked immunosorbent assay for detection of cytolethal distending toxin which involved the consecutive addition of either solubilized CHO or HeLa membranes, antigen and antibody. This enzyme-linked immunosorbent assay consistently detected crude cytolethal distending toxin diluted up to 16-fold. The receptor-based enzyme-linked immunosorbent assay for detection of cytolethal distending toxin developed in this study is a suitable alternative assay which can be performed easily in laboratories with minimal facilities and, more importantly, the results are available within a few hours as compared to times of up to 5 days in the conventional tissue culture detection of cytolethal distending toxin.
Bag
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Department of Microbiology, National Institute of Cholera and Enteric Diseases, Beliaghata, Calcutta, India.
Ramamurthy
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T
Nair
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UB
D000818:Animals; D001427:Bacterial Toxins / Q000302:isolation & purification / Q000378:metabolism*; D016466:CHO Cells / Q000378:metabolism; D016123:Campylobacter jejuni; D002462:Cell Membrane / Q000378:metabolism; D002772:Cholera Toxin / Q000378:metabolism; D006224:Cricetinae; D004768:Enterotoxins / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D029968:Escherichia coli Proteins; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D011956:Receptors, Cell Surface / Q000378:metabolism*
D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D011956:Receptors, Cell Surface; C067044:cytolethal distending toxin; D002772:Cholera Toxin; C027538:heat-labile enterotoxin, E coli