[Measurement of peroxidase activity with orthophenylenediamine as chromogen in immunoenzyme methods]

The authors studied orthophenylenediamine in order to determine the optimal conditions for its use in "sandwich" type of enzymo-immunology. They studied the kinetics of the reaction and the stopping of the enzymatic reaction in the presence of peroxidase-labeled antibody. Studies of the dissolution of orthophenylenediamine and the stability of the solutions obtained show that a solution of orthophenylenediamine can be stored at ambiant temperature and in darkness for 8 hours. Depending on the degree of sensitivity required, the concentration of orthophenylenediamine can vary between 10 to 30 mmol/l with 10 mmol/l of H2O2. Stopping the reaction with acid greatly increases the values of absorbance by 1.5 to 3 times, depending on the type of acid used. The tests demonstrated that the sensitivity and repeatability are better than can be obtained with 2,2' azino-di(3-ethylbenzthiazoline-6-sulfonic) acid. On the basis of these experiments, orthophenylenediamine was used in the assay of ferritin and total IgE: the increased sensitivity meant that the amount of antigen introduced into the reaction could be reduced to 1/5 of the amount usually required.