Highly sensitive determination of DAO activity by oxidation of a luminescence reagent

A highly sensitive method for measuring the activity of the enzyme diamine oxidase (DAO) independent of the type of substrate is described. The principle of the assay is to determine the amount hydrogen peroxide generated as a reaction product during oxidation of diamines by DAO. PSatto, a highly sensitive luminescence reagent, was used to generate a signal depending on the hydrogen peroxide concentration based on the action of horseradish peroxidase. DAO is specifically captured from a sample by an antibody immobilized to microwell plates, and the substrate is added to the bound enzyme. Various diamines were used as substrates; the peroxide produced is directly proportional to the amount of DAO bound to the specific antibodies. With this very sensitive method, it is possible to detect pmol amounts of generated hydrogen peroxide in plasma matrix corresponding to the biological activity of DAO.