Clinical Chemistry
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Clinical Chemistry 47: 569-574, 2001;
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(Clinical Chemistry. 2001;47:569-574.)
© 2001 American Association for Clinical Chemistry, Inc.

Articles

Homogeneous Enzyme Immunoassay for Triiodothyronine in Serum

Christina D. Karapitta1,2, Theodore G. Sotiroudis1,a, Athanassios Papadimitriou3 and Aristotelis Xenakis1

1 Industrial Enzymology Unit, Institute of Biological Research & Biotechnology, The National Hellenic Research Foundation, 48 Vassileos Constantinou Ave., 11635 Athens, Greece.

2 MEDICON S.A., 15344 Gerakas, Greece.

3 Department of Nuclear Medicine, Navy’s Hospital, 11521 Athens, Greece.
a Author for correspondence. Fax 30-1-7273758; e-mail tsotir{at}eie.gr.

Background: The concentration of triiodothyronine (T3) in human serum is extremely low and can be determined only by very sensitive methods. We developed a homogeneous enzyme immunoassay for T3 analysis in unextracted serum.

Methods: A T3 derivative was conjugated to the -SH groups of glycogen phosphorylase b (GPb) from rabbit muscle. Conjugation caused inhibition of enzyme activity, and the enzyme conjugate was reactivated upon binding of anti-T3 antibody. Activation was blocked by the presence of non-antibody-bound T3; this was the basis for the development of the homogeneous enzyme immunoassay for T3 by determining GPb activity fluorometrically.

Results: We used furosemide to block the interaction of T3 with serum proteins with T3-binding sites, avoiding any serum treatment step. T3 was measured in the range 0.3–8 µg/L. T3 values obtained by this assay correlated well with those obtained by a RIA (y = 0.97x - 0.07 µg/L; r = 0.96; n = 92). Within- and between-run imprecision (CV) was 5–9% for normal and high concentrations and 16–20% for low concentrations.

Conclusions: Chemical modification of GPb with a T3 derivative allows the development of a simple homogeneous enzyme immunoassay for T3 in unextracted serum.






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