Stabilization of the Substrate Reaction of Horseradish Peroxidase with o-Phenylenediamine in the Enzyme Immunoassay

Abstract

Summary: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 ilattening of the Standard curve in the enzyme immunoassay, after the reaction has been stopped. Addition of reducing agents, such äs sulphite ions, to the stopping solution, prevents the further oxidation of 0-phenylenediamine by completely reducing the remaining hydrogen peroxide. The developed colour is then stabilized. Stabilisierung der Substratreaktion mit o-Phenylendiamin und Wasserstoffperoxid für Meerrettich-Peroxidase im EnzymimmunoassayZusammenfassung: Nach Abbruch der Peroxidasereaktion durch Säurezugabe führt der Zerfall von nicht umgesetztem Wasserstoffperoxid zur weiteren Oxidation von o-Phenylendiamin, was zu einer Abflachung der Standardkurven im Enzymimmunoassay mit zunehmender Zeit nach Reaktionsabbruch führt. Der Zusatz von Reduktionsmitteln wie beispielsweise Sulfitionen zur Stopplösung verhindert durch Reduktion des verbliebenen Wasserstoffperoxids die weitere Oxidation von 0-Phenylendiamin und stabilisiert die entwickelte Farbe. Introduction immunoassays (EIA).A disadvantage of o-phe-0-Phenylenediamine is one of the most frequently nylenediamine, however, is its instability. It is thereused chromogens for the determination of horseradish fore advisable to perform the reaction in the dark peroxidase. Under optimal reaction conditions a de-and to measure the colour intensity of the developed tection limit of about 2 ng/1 horseradish peroxidase 2.2 diamino azobenzene immediately after stopping is observed (1), which enables highly sensitive enzyme the reaction by addition of acid (2).

The Characteristic of an Anti-Human DR5 Antibody A6

“…The reaction was terminated by 1 M H 2 SO 4 . The absorbance was measured chromatically at 492 nm with an ELISA-reader (Anthos, Austria) (16).…”

Novel chimeric anti-ricin antibody C4C13 with neutralizing activity against ricin toxicity

“…The reaction was terminated by 1 M H 2 SO 4 . The absorbance was measured at 492 nm with an ELISA-reader (Anthos, Austria) (Porstmann et al 1985).…”

Glutamic acid-141: a heme ‘bodyguard’ in anionic tobacco peroxidase

“…Iodide ion: 1.5 mM potassium iodide, 5 mM H 2 O 2 in 0.1 M Na-acetate buffer, pH 5.25;´3 53 s26 000 M -1 cm -1 (Bhattacharyya et al, 1992). o-Phenylenediamine: 1 mM o-PD, 1 mM H 2 O 2 in 0.1 M Na-acetate buffer, pH 5.0;´4 45 s11 100 M -1 cm -1 (Gallati and Brodbeck, 1982;Porstmann et al, 1985). o-Phenylenediamine: 1 mM o-PD, 1 mM H 2 O 2 in 0.1 M Na-acetate buffer, pH 5.0;´4 45 s11 100 M -1 cm -1 (Gallati and Brodbeck, 1982;Porstmann et al, 1985).…”