The principle of the competition assay is an indirect ELISA in which the soluble target is coated on a 96-well plate. After a blocking step, the samples are incubated simultaneously with a constant concentration of biotinylated ligand, both competing for binding to the coated target. HRP-labelled streptavidin subsequently binds to the biotinylated ligand but not to the unlabeled sample. 3,3,5,5-Tetramethylbenzidine (TMB) is converted by the HRP, resulting in a color signal that is inverse proportional to the sample concentration. In the final step, this reaction is stopped by acidification and the optical density (OD) of each well is measured using the ELISA microplate reader SpectraMax.