APPLICATION OF ORTHO-PHENYLENEDIAMINE AS A FLUOROGENIC SUBSTRATE IN PEROXIDASE-MEDIATED ENZYME-LINKED-IMMUNOSORBENT-ASSAY

o-Phenylenediamine (o-PD) is one of the most effective substrates for spectrophotometric horseradish peroxidase (HRP)-mediated enzyme-linked immunosorbent assay (ELISA), giving 2,3-diaminophenazine (DAP) as a product of o-PD oxidation. In this study it was found that the fluorescence of DAP in aqueous buffer solutions is considerably increased on adding a detergent (Triton X-100). DAP embedded in the micelles of Triton X-100 has absorption and fluorescence wavelength maxima at 428 and 554 nm, respectively, with a fluorescence quantum yield of 0.09. These properties make it possible to use high-purity o-PD as a fluorogenic substrate for HRP-mediated ELISA. The limit of detection of HRP by the spectrofluorimetric method in buffer solutions is 0.56-mu-U ml-1. The applicability of the proposed technique is demonstrated by the determination of carcinoembryonic antigen.