A novel method for time-resolved fluorimetric determination and imaging of the activity of peroxidase, and its application to an enzyme-linked immunosorbent assay

A new type of fluorescence assay for the determination of peroxidase (POx) activity is presented. The assay is based on the indication of the enzymatic consumption of H(2)O(2) (HP), using a fluorescent europium-tetracycline (Eu(3)TC) complex as indicator. On addition of HP, this complex forms a highly fluorescent adduct (Eu(3)TC-HP), which is decomposed in the presence of POx to form the weakly fluorescent europium-tetracycline (Eu(3)TC). Hence, the activity of the enzyme can be directly determined by means of the luminescent Eu(3)TC complex as indicator. The POx assay demonstrated herein was elaborated starting from a spectral characterization of the complex systems involved. Due to the long lifetime of lanthanide luminescence, both steady-state and time-resolved luminescence assays can easily be performed. The time-resolved assay can quantify POx in the range from 4.0 x 10(-5) to 5.9 x 10(-3) U mL(-1), with a limit of detection of 1.0 x 10(-5) U mL(-1). The effects of POx inhibitors such as cyanide, hydroxylamine, and azide have also been studied. In addition, a time-resolved fluorescent detection method for a POx-based enzyme-linked immunosorbent assay (ELISA) has been developed, which is demonstrated in a sandwich model assay with bovine IgG serving as analyte. Furthermore, a time-resolved fluorescent imaging method is demonstrated that makes use of a straightforward imaging set-up adjusted to the optical properties of the europium reagent.