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

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 mug/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 + 1.0x, 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. (C) 2001 Elsevier Science B.V. All rights reserved.