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

[corrected] 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.

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).

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 microg/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).

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.