A general fluorogenic assay for catalysis using antibody sensors

A general assay for monitoring catalysis by fluorescence in real time has b een developed by use of an antibody sensor. The sensor consists of a produc t-specific antibody tightly bound to a product analogue covalently labeled with the fluorescent tag acridone. Acridone fluorescence is quenched in the bound state. The reaction is monitored by following the fluorescence incre ase caused by displacement of the acridone-labeled product from the antibod y combining site by the released product. Fluorescence detection of enzymat ic hydrolysis of a beta-galactoside and butyrate by beta-galactosidase and esterase, respectively, are demonstrated. The assay operates by modulation of fluorescence intensity at 445 nm, a signal compatible with currently ava ilable instruments measuring in 96-well or 384-well plastic microtiter plat es. The method is potentially general and only limited by binding selectivi ties of the product versus the substrate that can be encountered in antibod ies.