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A new homogeneous enzyme immunoassay for thyroxine using glycogen phosphorylase b-thyroxine conjugates
C D Karapitta1, A Xenakis, A Papadimitriou
1Industrial Enzymology Unit, Institute of Biological Research and Biotechnology, The National Hellenic Research Foundation, 48 Vassileos Constantinou Ave., 11635, Athens, Greece.
Background:
[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.
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 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).
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.