Chemiluminescent determination of the activity of caspase-3 using a specific peptide substrate and magnetic beads

We report on a new scheme for the determination of the activity of caspase-3 using a specific peptide labeled with N-(4-aminobutyl)-N-ethylisoluminol (ABEI) as a chemiluminescent (CL) probe and on the development of magnetic separation technology. Firstly, the ABEI-labeled and biotinylated peptide was prepared and conjugated to streptavidin-coated magnetic beads (MBs) to form f-MBs (functionalized magnetic beads). The f-MBs contain a site (DEVD, Asp-Glu-Val-Asp) that is cleaved by caspase-3. Upon cleavage, the terminal residue attached to ABEI can dissociate from the f-MBs and can be used for CL detection. CL intensity is linearly related to the concentration of caspase-3 in the range 1.0 to 600 ng mL(-1), with a detection limit of 0.3 ng mL(-1). The relative standard deviation of the assay is 3.6 % at a level of 50 ng mL(-1) of caspase-3 (for n = 11). The CL assay has been applied to the determination of caspase-3 in Jurkat cell extract with recoveries between 96.6 % and 106.1 % (n = 5).