The Leptin ELISA is a solid phase enzyme-linked immunosorbent assay (ELISA) based on the sandwich principle.

The microtiter wells are coated with a monoclonal antibody directed towards a unique antigenic site of the leptin molecule. During the first incubation, leptin in the added sample binds to the immobilized antibody. Unbound sample material is washed off.

In a second incubation added antiserum, which contains biotinylated anti-leptin antibody, forms a sandwich complex with the bound leptin on the microtiter wells. Afterwards, the unbound antiserum is washed off and streptavidin peroxidase complex (Enzyme Complex) is added for detection of the biotin molecules.

After a washing step to remove all unbound substances, the solid phase is incubated with the substrate solution.

The colorimetric reaction is stopped by addition of stop solution, and optical density (OD) of the resulting yellow product is measured. The intensity of color is proportional to the concentration of the analyte in the sample. A calibration curve is constructed by plotting OD values against concentrations of calibrators, and concentrations of unknown samples are determined using this calibration curve.