17-Beta Estradiol (E2), ELISA, 96-test
The 17-Beta Estradiol (E2), ELISA is an immunoassay for the quantitative and sensitive detection of 17-Beta Estradiol. Kit Principle: Competitive Reaction The test is based on the recognition of E2 by specific monoclonal antibodies. E2 present in the sample and an E2-enzyme conjugate (i.e. E2 labeled with a coloring enzyme:HRP) are premixed and added into each well of a microplate, and allowed to compete for limited number of binding sites of specific antibodies immobilized on the surface of the wells. When the E2 concentration is higher relative to the enzyme conjugate, the E2 will predominantly bind the antibody and vice versa. Chromogenic Reaction Unbound E2 and excess E2-enzyme conjugates are washed out. The presence of E2 is detected by adding a chromogenic substrate:TMB. The enzyme-labeled E2 bound to the E2 antibody in the plate, catalyzes the conversion of the substrate to a colored product. After an incubation period, the reaction is stopped by the addition of a diluted acid. The higher the E2 concentration in a sample, for example, leads to less antigen-enzyme conjugate bound to the antibody binding sites in a microplate well, generating a lighter color, i.e. lower absorbance. Quantitative Analysis The standard curve, a dose-response curve obtained from known concentrations of E2 standards, is determined from the absorbance at 450nm. The E2 concentration in each sample is accurately calculated by interpolation using the absorbance intensity obtained from the standard curve. Features -17β-estradiol (E2) monoclonal antibody binds exclusively with E2 and does not show cross-reaction with other chemicals of similar structures. A monoclonal antibody is uniform in quality, generating very little lot-to-lot variation. -The quantitative analysis ranges from 0.05μg/L to 1μg/L (ppb). -The ELISA measurement is highly reproducible; the coefficient of variation (CV) is mostly under 10%. -The assay requires less amount of harmful solvent than instrument analyses. -With ease of handling, the total time for measurement is only 2.5 hours. -The kit, a 96-well microplate format, enables simultaneous measure- ment of multiple samples at more reasonable cost.