Development of an ADP-ribosylation assay for residual toxicity in C. difficile binary toxin CDTa using automated capillary western blot

Abstract

CDTa, an actin ADP-ribosylation transferase, is a binary toxin produced by the bacterium Clostridium difficile which is commonly associated with the hypervirulent strain present in Clostridium difficile infections. The mutated form of CDTa, 4mCDTa, is one of the components in the tetravalent Clostridium difficile vaccine in which the residual toxicity of the ADP-ribosylation activity needs to be monitored for safety reasons. There are several ADP- ribosylation activity methods employing techniques such as ELISA, manual Western blot, or SDS PAGE, but all these methods are usually time consuming and labor intensive. Here we describe the development of new quantitative capillary based western for monitoring the presence of ADP-ribosylation activity in CDTa and 4mCDTa using novel, automated Simple Western™ technology. Furthermore, we have measured for the first time the enzyme’s kinetic parameters, KM (NAD) and kcat for native CDTa using this new quantitative capillary western technology.