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The FSH ELISA is a two-step capture or ‘sandwich’ type immunoassay. The assay makes use of two highly specific monoclonal antibodies: A monoclonal antibody specific for FSH is immobilized onto the microplate and another monoclonal antibody specific for a different epitope of FSH is conjugated to horse radish peroxidase (HRP conjugate). In the first incubation step, FSH present in the specimen samples, calibrators and controls is bound by the antibody immobilized onto the microplate. Excess and unbound materials are removed by a washing step. In the second incubation step, HRP conjugate antibody (HRP conjugate) is added, which binds specifically to any immobilized FSH, thus forming a sandwich complex. Unbound HRP conjugate is removed by a washing step. Next, the TMB substrate (enzyme substrate) is added which reacts with HRP to form a blue coloured product that is directly proportional to the amount of FSH present. The enzymatic reaction is terminated by the addition of the stopping solution, converting the colour from blue to yellow. The absorbance is measured on a microplate reader at 450 nm. A set of calibrators is used to plot a calibrator curve from which the amount of FSH in specimen samples and controls can be directly read.