A Sensitive, Cell-Based Assay for Measuring Low-Level Biological Activity of α-Amanitin

alpha-Amanitin is one of the primary toxins produced by the poisonous mushroom genus, Amanita. Because it is odorless and tasteless, it is an important cause of death from the consumption of misidentified mushrooms. To study the thermal stability of alpha-amanitin, novel cell-based assays were developed to measure the toxin's activity, based on the inhibition of RNA polymerase II by alpha-amanitin. First, an MTT-formazan cell viability assay was used to measure the biological activity of alpha-amanitin through the inhibition of cellular activity. This method can detect 10 mu g/mL of alpha-amanitin in a time-dependent manner. Second, a more sensitive quantitative PCR approach was developed to examine its inhibition of viral replication. The new RT-qPCR assay enabled the detection of 100 ng/mL. At this level, alpha-amanitin still significantly reduced adenovirus transcription. Third, a simpler GFP expression-based assay was developed with an equal sensitivity to the RT-qPCR assay. With this assay, aqueous alpha-amanitin heated at 90 degrees C for 16 h or treated in the microwave for 3 min retained its biological activity when tested in HEK293 cells, but a slight reduction was observed when tested in Vero cells. Beyond detecting the activity of alpha-amanitin, the new method has a potential application for detecting the activity of other toxins that are RNA polymerase inhibitors.