Quantification of active caspase 3 in apoptotic cells

We describe an enzyme-linked immunosorbent assay (ELISA) for quantifying re lative amounts of active caspase 3 in apoptotic cells. Covalent modificatio n of caspase 3 active sites with a biotinylated inhibitor differentiates ac tive from latent caspases. Capture on an ELISA plate with an antibody speci fic for caspase 3 makes the assay specific for caspase 3. Detection is with horseradish peroxidase (HRP)-conjugated streptavidin that binds to the bio tinylated inhibitor covalently bound to caspase 3. Using the assay we detec ted 6.6 ng active caspase 3 per 10(6) apoptotic staurosporine-treated Jurka t cells. Specificity of the assay for caspase 3 was demonstrated by lack of signal with purified caspases 2, 7, 8, and 10 that were modified by a biot inylated inhibitor. Specificity was also demonstrated by lack of signal wit h apoptotic MCF-7 cells which do not express caspase 3. The ability to disc riminate between active and latent caspase 3 was shown by Western blotting with HRP-streptavidin and anti-caspase 3. Although latent caspase 3 was cap tured it was not covalently modified with the biotinylated inhibitor. The b asic principle of using a covalent inhibitor to identify active enzymes and an antibody to differentiate between enzymes with similar activities has p otential for quantifying active members of many classes of enzymes. (C) 200 0 Academic Press.