A new homogeneous enzyme immunoassay for thyroxine using glycogen phosphorylase b–thyroxine conjugates

Abstract

Background: Measurement of serum thyroxine (T 4) concentration is important for diagnosis of thyroid gland diseases. We developed a practical homogeneous enzyme immunoassay for thyroxine analysis in unextracted sera. Methods: A thyroxine derivative conjugated to a reactive sulfhydryl group of glycogen phosphorylase b (GPb). Conjugation caused inhibition of enzyme activity and the enzyme conjugate was re-activated upon the binding of a polyclonal anti-T 4 antibody. Antibody-activation was blocked by the presence of free T 4. Results: Conjugation affected the allosteric character of the enzyme and the K m for the allosteric activator AMP was increased 28 times, while anti-T 4 antibody partially reversed this effect. The optimum concentration ratio of enzyme conjugate to anti-T 4 antibody was determined, and T 4 was measured with desired sensitivity and accuracy in the range between 10 and 240 μg/l. Furosemide was used to inhibit the interaction of thyroxine with serum T 4-binding sites. Human serum T 4 values obtained by this method correlated well with those obtained by a radioimmunoassay ( y=1.9+1.0 x, r=0.97, N=72). Conclusions: Chemical modification of glycogen phosphorylase b with a T 4 derivative led to the development of a simple homogenous enzyme immunoassay for T 4 analysis with the desired sensitivity and accuracy.