Oxidation absorbance changes’ phases during

Determination of POase activity in solid-phase EIA is different from that of free POase in solution (Section 10.1.1.5). The amount of chromophore produced during a certain period (20 min, 30 min, 1 h, etc.) is determined from the absorbance [5-AS at 492 nm ABTS at 415 nm OPD at 492 nm if acidified with HCI (pH 1.0), at pH 5.0 the peak is at 445 nm (usual filter 436 nm) ODA at 400 nm DMAB/MBTH at 590 nm (usual filter, 620 nm)]. To obtain the exact color development in each sample, it is necessary to stop the reaction after identical periods. Color development with ABTS and 5-AS can be stopped with 0.2 volume of 37 mM NaCN (poison ), oxidation of OPD is blocked with 0.5 volume of 4 M H2SO4 or HCI and the reaction with MBTH/DMAB is arrested with 0.1 volume of IM H2SO4 or HCI (the blue-purple color changes to blue, but the absorption at 590 nm remains virtually unchanged). 

---