This assay is a two-site ELISA. The micro-plate is pre-coated with a monoclonal antibody against insulin. Standards and samples are added into the wells and co-incubated with a monoclonal antibody conjugated to horseradish peroxidase (HRP) enzyme. After wash step to remove any unbound substances, TMB substrate is added and colour develops in proportion to the amount of insulin bound initially. The assay is stopped and the optical density of the wells determined using a micro-plate reader. Since the increases in absorbance are directly proportional to the amount of captured insulin, the unknown sample concentration can be interpolated from a reference curve included in each assay.