In the Mouse and Rat Leptin BioAssay™ ELISA, standards, quality controls, and samples are incubated in microplate wells coated with anti-mouse leptin antibody. After 60 minutes incubation and washng, biotin-labeled polyclonal anti-mouse leptin antibody is added to the wells and incubated with immobilized antibody-leptin complex for 60 minutes. After another washing, Streptavidin-HRP conjugate is added. Ater 30 minutes incubation and the last washing step, the remaining conjugate is allowed to react with the substrate solution (TMB). The reaction is stopped by the addition of acidic solution and the absorbance of the resulting yellow product is measured spectrophotometrically at 450nm. The absorbance is proportional to the concentration of leptin. A standard curve is constructed by plotting the absorbance values versus the leptin concentrations of standards and concentrations of unknown samples are determined using this standard curve.