Homogeneous enzyme immunoassay for triiodothyronine in serum.
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.
Industrial Enzymology Unit, Institute of Biological Research & Biotechnology, The National Hellenic Research Foundation, 48 Vassileos Constantinou Ave., 11635 Athens, Greece.
C D
CD
Karapitta
T G
TG
Sotiroudis
A
A
Papadimitriou
A
A
Xenakis
D000328:Adult; D000368:Aged; D000818:Animals; D001665:Binding Sites; D001798:Blood Proteins / Q000378:metabolism; D005260:Female; D005470:Fluorometry; D005665:Furosemide; D006801:Humans; D007124:Immunoenzyme Techniques; D007202:Indicators and Reagents; D007527:Isoenzymes; D008297:Male; D008301:Maleimides; D008875:Middle Aged; D006005:Phosphorylases; D011817:Rabbits; D011863:Radioimmunoassay; D014284:Triiodothyronine / Q000097:blood* / Q000378:metabolism
D001798:Blood Proteins; D007202:Indicators and Reagents; D007527:Isoenzymes; D008301:Maleimides; D014284:Triiodothyronine; C025285:N-(4-carboxycyclohexylmethyl)maleimide N-hydroxysuccinimide ester; D005665:Furosemide; D006005:Phosphorylases