A microtiter plate assay for N-acetyl-beta-D-glucosaminidase using a fluorogenic substrate.

We describe a simple endpoint method for the determination of N-acetyl-beta-D-glucosaminidase (NAGase; EC 3.2.1.30). NAGase uses a fluorogenic substrate, 4-methylumbelliferyl-N-acetyl-beta-D-glucosaminide, at pH 4.6, liberating the fluorescent 4-methylumbelliferone. The method is reproducible and fast both at room temperature and at 37 degrees C. The procedure developed can be used, e.g., in the diagnosis of bovine subclinical mastitis, where elevated NAGase activities are found in raw milk samples. The assay procedure has a high capacity and high sensitivity and several hundred milk samples can be screened per hour using 96-well microtiter plates and an automated fluorescence reader. In addition to its use in mastitis diagnosis, the assay can be used in the diagnosis of some diseases of human origin.