New fluorimetric assay of horseradish peroxidase using sesamol as substrate and its application to EIA

Horseradish peroxidase (HRP) is generally used as a label enzyme in enzyme immunoassay (EIA). The procedure used for HRP detection in EIA is critical for sensitivity and precision. This paper describes a novel fluorimetric assay for horseradish peroxidase (HRP) using sesamol as substrate. The principle of the assay is as follow: sesamol (3,4-methylenedioxy phenol) is reacted enzymatically in the presence of hydrogen peroxide to produce dimeric sesamol. The dimer is fluorescent and can be detected sensitively at ex. 347 nm, em. 427 nm. The measurable range of HRP was 1.0×10-18 to 1.0×10-15 mol/assay, with a detection limit of 1.0×10-18 mol/assay. The coefficient of variation (CV, n=8) was examined at each point on the standard curve, with a mean CV percentage of 3.8%. This assay system was applied to thyroid stimulating hormone (TSH) EIA using HRP as the label enzyme.