4-Trifluoromethylumbelliferyl-β-D-Galactopyranoside: Its synthesis and Application as the Fluorigenic Substrate of β-GalactosidaseE. Colifor Screening Monoclonal Antibodies by Immunosorbent Elisa

Abstract

Abstract Stereoselective method for high-yield synthesis of 4-trifluoromethylumbelliferyl-β-D-galactopyrano-side - fluorigenic substrate for β-galactosidase E. coli, as enzyme label for ELISA, has been developed. The substrate is readily available in pure and stable form, nonfluorescent under the detection conditions and has no significant nonenzymatic degradation to fluorescent products. Compared to the well known 4-me-thylumbelliferone (λex = 360 nm, λem = 450 nm), 4-trifluoromethylumbelliferone has a slightly large Stokes shift (λex = 400 nm, λem = 500 nm), a longer excitation wavelength and emits green fluorescence through the enzymatic reaction. In the present ELISA we used 4-trifluoromethylumbelliferyl-β-D-galactopy-ranoside as sensitive enzyme substrate for rapid and visual screening of desired hybridoma clones producing monoclonal antibodies. With this immunoassay we succesfully selected antiperoxidase monoclonal antibodies which inhibited peroxidase activity.