Immunoassay of triiodothyronine in serum by time-resolved fluorometric measurement of europium-chelate complexes in solution.
We describe the development of a competitive immunoassay for triiodothyronine (T3) in serum. The assay combines immobilized antigens in microtitration wells with a biotinylated monoclonal anti-T3 antibody and a streptavidin-based universal detection reagent labeled with the Eu3+ chelator 4,7-bis(chloro-sulfophenyl)-1,10 phenanthroline-2,9-dicarboxylic acid (BCPDA). In the assay, T3 released from binding proteins by thimerosal competes with immobilized antigen for binding to a limited amount of antibody. The bound biotinylated antibody is identified by a subsequent reaction with the detection reagent, and fluorescence of the final complex is then quantified in solution after it has been dissociated from the solid support by the addition of a detergent solution. Evaluation of the method demonstrated good overall precision and appropriate detection limit (0.2 nmol/L) and dynamic range. Analytical recovery averaged 99.9%, and results of dilution experiments were in agreement with the expected values. Measurements by the present method correlated well with those by a commonly used radioimmunoassay.
Papanastasiou-Diamandi
A
A
CyberFluor Inc., Toronto, Ontario, Canada.
Shankaran
P
P
Khosravi
M J
MJ
D000911:Antibodies, Monoclonal; D005063:Europium; D005456:Fluorescent Dyes; D015200:Fluoroimmunoassay / Q000379:methods*; D006801:Humans; D010618:Phenanthrolines; D011863:Radioimmunoassay; D012680:Sensitivity and Specificity; D014284:Triiodothyronine / Q000097:blood* / Q000276:immunology
D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes; D010618:Phenanthrolines; D014284:Triiodothyronine; C054206:4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid; D005063:Europium