Design Of An ELISA Protocol For A Rapid Quantification Of Activated/oligomerized Bax During Apoptosis

Abstract

The release of cytochrome c from mitochondria into the cytosol is considered to be the commitment step of early apoptosis. During this step, the activation/oligomerization of the pro-apoptotic protein Bax leads to the formation of the Mitochondrial Apoptosis-Induced Channel (MAC) through which cytochrome can be released. Remarkably, Bax activation/oligomerization leads to the exposure of the previously inaccessible N-terminal domain of the pro-apoptotic protein Bax. This property was extensively used to detect activated/oligomerized Bax by immunocytochemistry and immunoprecipitation but these assays are long and tedious; and they are only qualitative or semi-quantitative at best.During this study, we developed an ELISA protocol based on the utilization of monoclonal (6A7) and polyclonal (N-20) antibodies which specifically recognize Bax N-terminus (i.e. the activated/oligomerized form of Bax). Monomeric human recombinant Bax (hBax) was used in all the experiments. As previously described, activation/oligomerization of hBax was artificially triggered by an exposure of the recombinant protein to the detergent Triton X-100. We first confirmed that the N-20 and 6A7 antibodies were able to fully immunoprecipitate the activated/oligomerized form but not the monomeric form of hBax. We then develop a sandwich ELISA using the 6A7 and the N-20 respectively as capture and detection antibodies. Reproducible standard curves were obtained when using increasing amounts of activated/oligomerized hBax. Monomeric hBax was never detected during these assays, even when previously mixed with activated/oligomerized hBax. Taken together, these results show that this ELISA allows a specific and quantitative detection of activated/oligomerized recombinant Bax. Finally, experiments of quantification of activated/oligomerized Bax with this technique in protein extracts from control and apoptotic cells are currently underway.Supported by NYU College of Dentistry Research Faculty Funds to LD.