1. The plate cells are coated with rabbit antibodies to estradiol.x
Stds and Unks compete with estradiol linked to horseradish peroxidase in enzyme conjugate for antibody binding sites. x
E-HP E
The bound estradiol peroxidase is measured by its reaction to Tetramethylbenzidine (TMB), producing a blue color. x
E-HP E
TMB
After stopping the Rx with sulfuric acid, color turns yellow. x
E-HP E
H2SO4
Spectroradiometric plate reader measures optical density of cells at 450 nm and at 630 nm for correction.
@ of estradiol peroxidase detected, which is inversely proportional to of estradiol present.