A new visual screening assay for catalytic antibodies with retro-aldol retro-Michael activity

Fast and convenient methods are required for the detection of novel catalysts. We have developed a new assay to allow direct visualization of retro-aldol retro-Michael catalytic activity and have demonstrated it with catalytic antibody 38C2. The assay is based on a catalytic cleavage of a physiologically stable substrate to release 3,4-cyclohexeneoesculetin. The latter then reacts with iron(III) to generate a non-soluble complex that precipitates in the form of a black dye. This assay may be used for screening new catalysts for retro-aldol retro-Michael activity with improved efficiency for specific prodrug activation.