Principle of Assay
Human Estradiol ELISA Kit (A327138) employs a rapid competitive Enzyme-Linked Immunosorbent Assay (ELISA) technique for the quantitative measurement of human Estradiol in serum, plasma, urine, and saliva. Antigen specific for Estradiol has been pre-coated onto a 96-well microtiter plate. Standards and test samples are added to the wells together with HRP-conjugated detection reagent working solution. During incubation, Estradiol present in the sample competes with the immobilized antigen for binding to the HRP-conjugated detection reagent. Following incubation, unbound components are removed by washing. TMB substrate solution is then added to visualize the HRP enzymatic reaction, producing a blue-coloured product that changes to yellow following addition of acidic stop solution. The intensity of the yellow colour is inversely proportional to the concentration of Estradiol present in the sample. The concentration of Estradiol is determined by measuring the O.D. absorbance at 450 nm using a microplate reader and comparing the values to the standard curve.
