Horseradish peroxidase chemiluminescence

The emission yield from the horseradish peroxidase (HRP)-catalyzed luminol oxidations can be kicreased as much as a thousandfold upon addition of substituted phenols, eg, -iodophenol, -phenylphenol, or 6-hydroxybenzothiazole (119). Enhanced chemiluminescence, as this phenomenon is termed, has been the basis for several very sensitive immunometric assays that surpass the sensitivity of radioassay (120) techniques and has also been developed for detection of nucleic acid probes ia dot-slot. Southern, and Northern blot formats (121). 

Chemiluminescence and bioluminescence are also used in immunoassays to detect conventional enzyme labels (eg, alkaline phosphatase, P-galactosidase, glucose oxidase, glucose 6-phosphate dehydrogenase, horseradish peroxidase, microperoxidase, xanthine oxidase). The enhanced chemiluminescence assay for horseradish peroxidase (luminol-peroxide-4-iodophenol detection reagent) and various chemiluminescence adamantyl 1,2-dioxetane aryl phosphate substrates, eg, (11) and (15) for alkaline phosphatase labels are in routine use in immunoassay analyzers and in Western blotting kits (261—266). 

Catalytic oxidation of isobutyraldehyde with air at 30—50°C gives isobutyric acid [79-31-2] ia 95% yield (5). Certain enzymes, such as horseradish peroxidase, cataly2e the reaction of isobutyraldehyde with molecular oxygen to form triplet-state acetone and formic acid with simultaneous chemiluminescence (6). 

A method of detecting herbicides is proposed the photosynthetic herbicides act by binding to Photosystem II (PS II), a multiunit chlorophyll-protein complex which plays a vital role in photosynthesis. The inhibition of PS II causes a reduced photoinduced production of hydrogen peroxide, which can be measured by a chemiluminescence reaction with luminol and the enzyme horseradish peroxidase (HRP). The sensing device proposed combines the production and detection of hydrogen peroxide in a single flow assay by combining all the individual steps in a compact, portable device that utilises micro-fluidic components. 

Immunodetection is performed by chemiluminescence (ECL , Amersham Pharmacia Biotech) using horseradish peroxidase-conjugated secondary antibodies (Amersham Pharmacia Biotech). 

Based on IgG-bearing beads, a chemiluminescent immuno-biochip has been also realized for the model detection of human IgG. Biotin-labeled antihuman IgG were used in a competitive assay, in conjunction with peroxidase labelled streptavidin59. In that case, the planar glassy carbon electrode served only as a support for the sensing layer since the light signal came from the biocatalytic activity of horseradish peroxidase. Free antigen could then be detected with a detection limit of 25 pg (108 molecules) and up to 15 ng. 

A.N. Diaz, F.G. Sanchez, M.C. Ramos, and M.C. Torijas, Horseradish peroxidase sol-gel immobilized for chemiluminescence measurements of alkaline-phosphatase. Sens. Actuat. B 82, 176-179 (2002). 

In the reaction of luminol, hydrogen peroxide, and horse radish peroxidase 122> the chemiluminescence intensity is proportional to the square of luminol radical concentration. The lifetime of these luminol radicals was found by ESR techniques to be about 10 sec. Titration studies revealed that luminol acts as two-electron donor during the reduction of a hydrogen peroxide-horseradish peroxidase complex. The enzyme is not involved in the reaction step leading directly to light emission. This step is formulated as... 

HTAC cationic micelles also markedly enhance the CL intensity of fluorescein (FL) in the oxidation of hydrogen peroxide catalyzed by horseradish peroxidase (HRP) [39], However, no CL enhancement was observed when anionic micelles of sodium dodecyl sulphate (SDS) or nonionic micelles of polyoxyethylene (23) dodecanol (Brij-35) were used (Fig. 9). CL enhancement is attributed to the electrostatic interaction of the anionic fluorescein with the HTAC micelles. The local concentration of fluorescein on the surface of the micelle increases the efficiency of the energy transferred from the singlet oxygen (which is produced in the peroxidation catalyzed by the HRP) to fluorescein. This chemiluminescent enhancement was applied to the determination of traces of hydrogen peroxide. The detection limit was three times smaller than that obtained in aqueous solution. 

ABEI, M(4-ami nobutyl )-Methylisolu mi nol BSA, bovine serum albumin CL, chemiluminescence DNPO, tas-(2,4-dinitrophenyl)oxalate ECL, electrogenerated chemiluminescence EMMA, electrophoretically mediated microanalysis EY, eosine Y FR, lluorescamine HRP, horseradish peroxidase ILITC, isoluminol isothiocyanate LOD, limit of detection RITC, rhodamine B isothiocyanate TCPO, Mv-(2,4,6-trichlorophenyl)oxalate TEA, triethylamine TRITC, tetramethylrhodamine isothiocyanate. 

Horseradish peroxidase (HRP)-coupled secondary anti-IgG antibodies (species depending on primary antibody) enhanced chemiluminescence substrate... 

Methods based on chemiluminescent and bioluminescent labels are another area of nonisotopic immunoassays that continue to undergo active research. Most common approaches in this category are the competitive binding chemiluminescence immunoassays and the immunochemiluminometric assays. Chemiluminescence and heterogenous chemiluminescence immunoassays have been the subject of excellent reviews (91, 92). Detection in chemiluminescence immunoassays is based on either the direct monitoring of conjugated labels, such as luminol or acridinium ester, or the enzyme-mediated formation of luminescent products. Preparation of various derivatives of acridinium esters has been reported (93, 94), whereas a variety of enzyme labels including firefly or bacterial luciferase (70), horseradish peroxidase (86, 98), and alkaline phosphatase are commercially available. 

Thorpe, G H. G and Kricka, L. J. (1986) Enhanced chemiluminescent reactions catalyzed by horseradish peroxidase. Methods Enzymol. 133, 331-354. 

Kricka, L. J., Stott, R A W., and Thorpe, G. H. G (1991) Enhanced chemiluminescent detection of horseradish peroxidase labels in ligand binder assays, in Luminescence Techniques in Chemical and Biochemical Analysis (Bayens, W R. G., De Kekeleire, D, and Korkidis, K, eds.), Dekker, New York, pp 599-635... 

Optical detection is possible when photoactive reagents were activated with the help of the enzyme label catalysis. For example, horseradish peroxidase could be used for chemiluminescence [32,33,35],... 

In glycoprotein detection systems the carbohydrate portions of proteins are oxidized with sodium metaperiodate to generate aldehydes that can react with hydrazides. A biotin hydrazide is used to attach biotin onto the oxidized carbohydrates and horseradish peroxidase-conjugated streptavidin is used for chemiluminescence-based detection (Glycoprotein Detection Module, Amersham Biosciences, Uppsala, Sweden). 

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