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Development of an ADP-ribosylation assay for residual toxicity in C. difficile binary toxin CDTa using automated capillary western blot.
Journal of Pharmaceutical and Biomedical Analysis ( IF 3.4 ) Pub Date : 2020-01-23 , DOI: 10.1016/j.jpba.2020.113125
Richard R Rustandi 1 , Melissa Hamm 1
Affiliation  

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.

中文翻译:

使用自动毛细管蛋白质印迹技术开发了用于艰难梭菌二元毒素CDTa残留毒性的ADP核糖基化测定法。

CDTa是肌动蛋白ADP-核糖基化转移酶,是艰难梭菌细菌产生的二元毒素,通常与艰难梭菌感染中存在的高毒力菌株有关。CDTa的突变形式为4mCDTa,是四价艰难梭菌疫苗中的一种成分,出于安全原因,其中ADP-核糖基化活性的残留毒性需要进行监测。有几种采用ELISA,人工Western印迹或SDS PAGE等技术的ADP核糖基化活性方法,但是所有这些方法通常都是费时且费力的。在这里,我们描述了基于新型定量毛细管的Western的开发,该监测用于使用新型自动化Simple Western™技术监测CDTa和4mCDTa中ADP核糖基化活性的存在。此外,
更新日期:2020-01-23
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