Stabilization of the substrate reaction of horseradish peroxidase with o-phenylenediamine in the enzyme immunoassay.

When the horseradish peroxidase reaction is stopped with acid, the decay of unconverted hydrogen peroxide is responsible for the further oxidation of o-phenylenediamine. This leads to a time-dependent flattening of the standard curve in the enzyme immunoassay, after the reaction has been stopped. Addition of reducing agents, such as sulphite ions, to the stopping solution, prevents the further oxidation of o-phenylenediamine by completely reducing the remaining hydrogen peroxide. The developed colour is then stabilized.