An investigation on the catalytic mechanism of enhanced chemiluminescence: immunochemical applications of this reaction.

The mechanism of peroxidase-catalysed oxidation of luminol by H2O2 was studied. The stopped-flow technique was used to measure the rate constants for the reactions between the oxidized forms of peroxidase with luminol and the following substrates: p-iodophenol, p-bromophenol, p-clorophenol, o-iodophenol, m-iodophenol, luciferin, and 2-iodo-6-hydroxybenzothiazole. The correlation between kinetic parameters and the degree of enhancement was established. The effect of charged synthetic polymers and specific antibodies on the peroxidase activity in the enhanced chemiluminescent reaction was also studied. The close approach of an effector molecule to the active site of the enzyme was found to inhibit the enhanced chemiluminescent reaction. Novel homogeneous methods of luminescent immunoassay (LIA) for (1) antibodies to insulin, (2) insulin and (3) antibodies to trinitrophenyl group are proposed on the basis of regulatory facilities of the enhanced chemiluminescent reaction. Based on the enhanced chemiluminescent reaction a peroxidase flow-injection assay was developed and successfully tested in the flow-injection enzyme immunoassays for human IgG and for thyroxin (T4). The immunoassay proposed has a detection limit of 10(-9) M for IgG and 10(-11) M for T4, the overall time of the assay being 5-15 min.