Illustration of the hook effect. This phenomenon arises because high concentrations of analyte saturate all antigen binding sites on the capture and label reagent antibodies and thereby interfere with sandwich-formation. A subsequent wash step removes all species not bound to the capture antibody (including analyte-label antibody complexes) and leads to a lower-than-expected signal during detection. (A) The analyte concentration is low relative to the number of available antibody binding sites. A hook effect does not occur. (B) The analyte concentration is high relative to the number of available antibody binding sites. The hook effect leads to falsely low signal.