A Fluorescent Peptide Substrate for Measuring the ADP-Ribosylation Activity of the Cholera Toxin A-Subunit

Abstract

Vibrio cholerae serogroups O1 and O139 Bengal produce cholera toxin (CT) a typical AB5 bacterial toxin comprising an ADP-ribosylation enzyme A-subunit (CTA) and a carbohydrate binding B-subunit (CTB). DUKORALR the inactivated oral cholera vaccine has recently been licensed for use in the European Union. This vaccine contains killed whole cells of V. cholerae and 1 mg of purified recombinant CTB (rCTB). DUKORAL^R has a good safety profile and there has been no indication that active CT is present. Nevertheless, an assay that confirms the absence of active CTA in the vaccine is advantageous to ensure vaccine safety. Conventional assays such as the Y-cell assay can not detect biologically active amounts of CT in DUKORAL^R because of the large amount of rCTB present. We have developed an assay based on a fluorescently labelled 11-mer peptide substrate that detects CTA activity despite the presence of excess rCTB.