Total Estrogens ELISA

The Calbiotech Inc., Total Estrogens ELISA kit is based on the principle of competitive binding between Estrogens in the test specimen and enzyme labeled estrogens for a constant amount of rabbit antiestrogens antibody. In the first incubation, goat anti-rabbit IgG-coated wells are incubated with 50μl of Total Estrogens standards, controls, patient samples, 100μl Estrogens-HRP conjugate reagent and 50μl rabbit anti-Total Estrogens reagent, at room temperature, for 120 minutes, with agitation. During the incubation, HRP labeled Total Estrogens competes with the endogenous estrogens in the standard, controls and sample, for a fixed number of binding sites of the specific anti Total Estrogens antibody. Thus, the amount of HRP labeled Estrogens conjugate immunologically bound to the antibody progressively decreases as the concentration of Total Estrogens in the specimen increases. Unbound Total estrogens enzyme conjugate is then removed and the wells washed. Then substrate (TMB) reagent is added and incubated, with agitation, for 30 minutes, resulting in the development of blue color. The color development is stopped with the addition of 50IL stop solution, and the absorbance is spectrophotometrically measured at 450nm. A standard curve is prepared relating color intensity to the concentration of Total Estrogens versus the absorbance.