Description
Principle of the assay: The MyBioSource Estrone-3-Sulfate equine ELISA test kit is a solid phase enzyme immunoassay (ELISA) in the microplate format, designed for the measurement of estrone-3-sulfate. The microplate is coated with a polyclonal antibody specific for estrone-3-sulfate. Calibrators and samples as well as a biotin-labeled estrone-3-sulfate are pipetted into the antibody coated microplate. During one hour incubation the estrone-3-sulfate of the calibrator/sample competes with the biotin-labeled estrone-sulfate for the binding sites of the antibody fixed on the inner surface of the wells. Afterwards, horseradish peroxidase-labeled streptavidine is added. During an 30 minutes incubation, this enzyme-labeled streptavidine binds to the biotinylated estrone-3-sulfate. All wells are washed to remove excess of non-bound enzyme-labeled streptavidine. A chromogenic substrate, TMB (3,3',5,5'-Tetra-Methyl-Benzidine), is added to all wells. During a 30 minutes incubation, the substrate is converted to a colored end product (blue) by the fixed enzyme. Enzyme reaction is stopped by dispensing of hydrochloric acid as stop solution (change from blue to yellow). The color intensity is inversely related to the concentration of estrone-3-sulfate present in the sample. The optical density of the color solution is measured with a microplate reader at 450 nm. Bi-chromatic measurement with a 600 - 690 nm reference filter is recommended