Fluorescent Enzyme Immunoassay of Free Thyroxin Using Glucose Oxidase as Enzyme Label

Abstract

A fluorescent enzyme immunoassay for free thyroxin using glucose oxidase as the label enzyme has been developed. Double antibody solid phase method was used. After separation of bound and free fractions the glucose oxidase activity was measured using a coupled enzyme procedure: glucose and horseradish peroxidase were used as the substrate and coupled enzyme, respectively. Hydrogen peroxide generated from glucose was determined by the fluorophotometric method using p-hydroxyphenylpropionic acid as a substrate. The proposed fluorescent enzyme immunoassay is uninfluenced by thyroxin-binding globulin or by albumin. Free thyroxin values in serum determined by the proposed method correlated well with those obtained by the equilibrium dialysis method (serum: r=0. 93). Free thyroxin in a dried blood filter paper disc (3 mm in diameter) could be assayed by this method. The mean variabilities (RSD) of within and between assays were 9. 6% and 6. 9% for sera and 7. 6% and 9. 1% for dried blood samples, respectively. The measurable range of free thyroxin was from 0. 05 to 5 ng/dl.