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

Abstract

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.

Rate constants for reactions of horseradish peroxidase compounds I and II with 4-substituted arylboronic acids

“…They suggest that because of the enhanced reactivity of 4-iodophenol, free radicals formed from it attack unreacted luminol, enhancing the production of luminol semiquinone radicals and thus enhancing chemiluminescence. O n the other hand, Vlasenko et al examine the possibility that the enhancer facilitates a higher steady concentration of HRP-I that in turn facilitates greater reactivity of luminol (11). They conclude that the latter possibility does not lead to a large enough enhancement.…”

Exchanging physically effective neutral detergent fiber does not affect chewing activity and performance of late-lactation dairy cows fed corn and sugarcane silages

“…Samples were collected in 7-mL vacuum tubes (Vacuette, Cen-Med Enterprises; New Brunswick, NJ) containing either no preservatives or K 3 EDTA-sodium fluoride for serum and plasma separation, respectively. After centrifugation (2,000 × g for 20 min at 5°C), insulin (chemiluminescence immunoassay; Vlasenko et al, 1989) was analyzed in the serum, whereas glucose (glucose oxidase; LABTEST Diagnóstica S.A., Lagoa Santa, Brazil; Trinder, 1969) and NEFA (colorimetric method; Randox Laboratories Ltd., Crumlin, United Kingdom; Johnson and Peters, 1993) were determined in the plasma.…”

Clinical applications of luminescent assays for enzymes and enzyme labels

“…Non-separation-enhanced chemiluminescent assays for insulin, insulin antibodies, and antibodies to the trinitrophenyl group can be produced using either polyclonal antiperoxidase antibodies or polycations such as poly-N-ethyl-4-vinylpyridinium bromide. The insulin immunoassay detected 2 X 10-llM in an 10 minute assay (6).…”