The Anti-Tg ELISA is a two-step capture or ‘sandwich’ type immunoassay. Human thyroglobulin (Tg) antigen is immobilized onto the microplate. In the first incubation step, anti-Tg autoantibodies present in the specimen samples, calibrators and controls is bound to the immobilized antigen. Excess and unbound materials are removed by a washing step. In the second incubation step, anti-human-IgG HRP conjugate is added, which binds specifically to any autoantibodies that are bound to the immobilized antigen. 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 anti-Tg autoantibodies present. The enzymatic reaction is terminated by the addition of the stopping solution, converting the blue colour to a yellow colour. 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 anti-Tg autoantibodies in specimen samples and controls can be directly read.