Chemiluminescence Enzyme Immunoassay for Thyroxin Using Glucose Oxidase and a Leuco-2', 7'-Dichlorofluorescein-Peroxidase-Hydrogen Peroxide-Bis (2, 4, 6-Trichlorophenyl) Oxalate System

Abstract

The new chemiluminescence assay of glucose oxidase (GOD) has been developed and applied to the enzyme immunoassay using GOD as the label enzyme. GOD activity can be assayed by two reaction steps in which at the first step non-fluorescent leuco-2', 7'- dichlorofluorescein is oxidized to fluorescent 2', 7'-dichlorofluorescein (DCF) by H₂O₂ generated from glucose with the catalytic reaction of GOD (to be assayed), and then at the second step DCF gives chemiluminescence by reaction with bis (2, 4, 6-trichlorophenyl) oxalate-H₂O₂ in acetonitrile. Detection limits of H₂O₂ and GOD were 10^-7 mol/l and 1.0μU/ml, respectively. This method was applied to the EIA of thyroxin (T₄) using GOD as the label. Results for T₄ by the EIA correlated well with those for serum determined by radioimmunoassay (r=0.947).