The principle of the adiponectin ELISA is a two-step sandwich enzyme immunoassay. The assay makes use of two highly specific monoclonal antibodies: A monoclonal antibody specific for adiponectin is immobilized onto the microplate and another monoclonal antibody specific for a different epitope of adiponectin is conjugated to biotin. During the first step, adiponectin present in the samples and standards is bound to the immobilized antibody and to the biotinylated antibody, thus forming a sandwich complex. Unbound biotinylated antibody is removed by a washing. In the second step, streptavidin-HRP is added, which binds specifically to bound biotinylated antibody. Unbound streptavidin-HRP is removed by washing. Next, the enzyme substrate (TMB) is added. The colour intensity of the enzymatic reaction is directly proportional to the concentration of adiponectin. The enzymatic reaction is terminated by the addition of stopping solution. The absorbance is measured on a microplate reader at 450 nm. The concentration of adiponectin in samples and controls can be calculated from of a plot of the standard curve, either graphically or by using immunoassay software.