Homogeneous enzyme immunoassay for triiodothyronine in serum

Background: The concentration of triiodothyronine (T-3) in human serum is extremely low and can be determined only by very sensitive methods. We developed a homogeneous enzyme immunoassay for T-3 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-T-3 antibody. Activation was blocked by the presence of non-antibody-bound T-3; this was the basis for the development of the homogeneous enzyme immunoassay for T-3 by determining GPb activity fluorometrically. Results: We used furosemide to block the interaction of T-3 with serum proteins with T-3-binding sites, avoiding any serum treatment step. T-3 was measured in the range 0.3-8 mug/L. T-3 values obtained by this assay correlated well with those obtained by a RIA (y = 0.97x - 0.07 mug/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 T-3 derivative allows the development of a simple homogeneous enzyme immunoassay for T-3 in unextracted serum. (C) 2001 American Association for Clinical Chemistry.