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Homogeneous enzyme immunoassay for triiodothyronine in serum
C D Karapitta1, T G Sotiroudis, A Papadimitriou
1Industrial Enzymology Unit, Institute of Biological Research & Biotechnology, The National Hellenic Research Foundation, 48 Vassileos Constantinou Ave., 11635 Athens, Greece.
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 T(3) 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 microg/L. T3 values obtained by this assay correlated well with those obtained by a RIA (y = 0.97x - 0.07 microg/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.