The first step of this assay is the adsorption of the antigen on the plate. With an overnight incubation a better immobilization could be determined. The signal after 1.5 h was good (OD 0.428 ± 0.042), however the signal increased by ∼30% with an overnight incubation (OD 0.637 ± 0.022) probably due to better adsorption of the antigen to the plate surface and was therefore chosen for the assay. To achieve the desired intensity of color from the peroxidase reaction, the optimum pH of the blocking buffer used for the dilution of the antibody was determined. This guarantees the highest activity of the conjugated peroxidase and thus results in an increase of the signal–noise ratio of the antibody. At pH 6.5 the peroxidase reaction developed a good signal in fast time and was therefore selected as optimum for the substrate phosphorylation detection