Horseradish peroxidase assay

Fluorescence-enzyme-linked immunosorbent assay. Horseradish peroxidase. 

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). 

Other specific discovery assays have been used such as differential inhibition of a vancomycin resistant S. aureus strain and its susceptible parent, and an assay based on antagonism of the antibacterial activity by N,A/-diacetyl-L-Lys-D-Ala-D-Ala [24570-39-6] a tripeptide analogue of the dalbaheptides receptor. AppHcation of this latter test to 1936 cultures (90) led to the isolation of 42 dalbaheptides, six of which, including kibdelin (Table 3), parvodicin (Table 3), and actinoidin A2 (68) were novel. A colorimetric assay based on competition between horseradish peroxidase bound teicoplanin and the... 

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. 

The sensitivity of enzyme assays can also be exploited to detect proteins that lack catalytic activity. Enzyme-linked immunoassays (ELlSAs) use antibodies covalently finked to a reporter enzyme such as alkafine phosphatase or horseradish peroxidase, enzymes whose products are readily detected. When serum or other samples to be tested are placed in a plastic microtiter plate, the proteins adhere to the plastic surface and are immobilized. Any remaining absorbing areas of the well are then blocked by adding a nonantigenic protein such as bovine serum albumin. A solution of antibody covalently linked to a reporter enzyme is then added. The antibodies adhere to the immobilized antigen and these are themselves immobilized. Excess free antibody molecules are then removed by washing. The presence and quantity of bound antibody are then determined by adding the substrate for the reporter enzyme. 

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. 

The major enzymes used in ELISA technology include horseradish peroxidase (HRP), alkaline phosphatase (AP), (3-galactosidase (P-gal), and glucose oxidase (GO). See Chapter 26 for a detailed description of enzyme properties and activities. HRP is by far the most popular enzyme used in antibody-enzyme conjugates. One survey of enzyme use stated that HRP is incorporated in about 80 percent of all antibody conjugates, most of them utilized in diagnostic assay systems. 

Fig. 8. Schematic presentation of a enzyme linked immunosorbent assay (ELISA). An antigen ( ) is immobilized on the surface of a microtiter plate and incubated with its antibody (abl). A second antibody (ab2) with a covalently linked enzyme ( , e.g., horseradish peroxidase) binds to the primary one and catalyzes a color reaction with its enzyme. All incubations are separated by washing steps...

Horseradish peroxidase (HRP) is an extracellular plant enzyme that acts in regulation of cell growth and differentiation, polymerization of cell wall components, and the oxidation of secondary metabolites essential for important pathogenic defense reactions. Because of these essential functions, and also because of its stability and ready availability, HRP has attracted considerable attention.13 It has been involved in a number of applications, such as diagnostic assays,14 biosensors,15 bioremediation,16 polymer synthesis,17 and other biotechnological processes.18 More applications in which HRP catalysis is translated into an electrochemical signal are likely to be developed in the near future. 

Enzymes can be linked to immunoassay reagents to amplify detection by the use of fluorogenic substrates. Enzyme-linked fluoroimmunoassays (ELFIAs) are very similar to photometric EIAs in format and workflow. EIAs are widely used, and many commercial ELFIA assays and systems are available/15 The most commonly used enzymes in ELFIAs are horseradish peroxidase, alkaline phosphatase, and fi-D-... 

A horseradish peroxidase-osmium redox polymer-modified glassy carbon electrode (HRP-GCE) has also been applied to this analysis to improve sensitivity and reduce problems with faradic interference. Kato and colleagues (1996) employed this electrode in measurement of basal ACh in microdialysates using a precolumn enzyme reactor. This system was three to five times more sensitive than a conventional Pt electrode. ACh in rat hippocampus dialysate was quantitated at 9 5 fmol/15 pi (n = 8). ACh was analyzed in PC12 cells in a similar assay by Kim and colleagues (2004). No precolumn enzyme reactor was employed. 

Probes tagged with an enzyme signal generating system, such as alkaline phosphatase or horseradish peroxidase, are sometimes used in direct assays. Alternatively, enzymes as reporters may be used with biotinylated probes in a direct affinity assay or in an indirect affinity assay. For the former assay, the... 

Double-antibody sandwich. Antibody is coated on or adsorbed to the plastic plate. The sample to be assayed containing the antigen (antibiotic) is added followed by a second antibody that is conjugated to the enz)rme (horseradish peroxidase, alkaline phosphatase, or 3 galactosidase). The substrate is added and the intensity of the color produced is directly proportional to the antigen in the test sample. 

Fig. 1. A simplified flow chart for dual-color bright-field in situ hybridization (BISH) assays using horseradish peroxidase (HRP) and/or alkaline phosphatase (AP)-based immunological signal detections.

Fig. 8. Morphological changes of apoptotic eosinophils induced by dexamethasone (Z2). After eosinophils were treated (a) without or (b) with dexamethasone (2 /u,M) for 12 h, cells were harvested and detected by TUNEL assay using the In Situ Cell Death Detection Kit (Boehringer Mannheim). Briefly, cells were fixed with 4% paraformaldehyde and permeabilized by proteinase K and incubated with the TUNEL reaction mixture containing terminal deoxynucleotidyl transferase (TdT). After washing to remove unbound enzyme conjugated antibody, the horseradish peroxidase retained in the immune complex was visualized by a substrate reaction with diaminobenzidine. The cell nucleus was counterstained with methanol green. Apoptotic eosinophils with nuclear DNA breaks were seen to stain dark brown using a Nikon Eclipse E800 microscope (Nikon Corporation, Tokyo, Japan) in Fig. 8b.

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