MODULATION OF THE ENHANCED CHEMILUMINESCENCE WITH ANTI-PEROXIDASE SPECIFIC ANTIBODIES

摘要

The effective application of horseradish peroxidase (HRP) in analytical biochemistry and biotechnology depends on its stability, especially when it is used repeatedly or in lengthy processes. HRP was shown to undergo irreversible time-dependent inactivation when catalysing different oxidation reactions. The peroxidation of some donor substrates results in the formation of highly reactive radical intermediates, which can in turn attack and modify the prosthetic group or apo-protein causing the loss of enzymatic activity. The enhanced chemiluminescence reaction (ECL) is used as a highly sensitive detection system for different assays. Recently we showed that the enzyme inactivation is the main reason for light decay, and highly reactive endogenous intermediates formed during enhancer oxidation were suggested as the main inactivating agents. As radical species modify the protein, our approach for HRP stabilization consists of a search for substances, which can form stable complexes with the enzyme. We revealed the stabilization of the light output when the ECL reaction was catalysed by HRP in complexes with linear polycations. The aim of the present work was to study the effect of monoclonal antibodies (mc Ab) specific to HRP on its stability during p-iodophenol oxidation and light emission in the ECL reaction.