Peroxidase chromogens

Enzymatic methods are commonly used for this measurement, and many of the current methods use a cholesterol esterase to hydrolyze lipoprotein-cholesteryl esters, followed by further reactions where cholesterol oxidase is linked to a peroxidase-chromogen system (Richmond 1992). Eree cholesterol can be measured by omitting cholesterol esterase of the first reaction step, although many reagent formulations prevent this elimination of the esterase. 

Niewola et al. [183, 185] have described a rapid, convenient and accurate method, based upon an enzyme-based immunosorbent assay (ELISA) for the determination of Paraquat residues in soil. Polystyrene plates, coated with paraquat-keyhole limpet haemocyanin (KLH) conjugate, are incubated with the test samples and a known amount of monoclonal antibody. Residual antibody that has not reacted with free Paraquat in the sample combines with paraquat-KLH on the plate. The determination of the fixed antibody is achieved by the addition of peroxidase labelled rabbit antimouse immunoglobulin G followed by reaction with a chromogenic substrate. The enzyme activity of the solid phase is determined from the absorbance measurements, which are inversely proportional to the concentration of Paraquat. The method shows high specificity and correlates well with the traditional ion exchange-spectrophotometric method for the determination of Paraquat [178]. 

The enzyme or the chromogen detection system determines whether any endogenous material must first be destroyed. If a peroxidase marker molecule is to be used, endogenous peroxidase or peroxidase-like activity should be blocked. Because these preparations are more fragile than a fixed embedded sample, endogenous enzyme is inactivated with a weaker blocking solution than would... 

There are a few common enzymes that have been employed in these types of assay systems over the years, the chief among them being the peroxidase enzyme (3). Peroxidase has an oxidative function when in conjunction with a source of oxygen, transferring electrons to a molecule, which becomes oxidized. The peroxidase enzyme found in the horseradish plant has been used for its ability to carry out this function, for the fact that it is easily obtained, and for the antigenic differences from most mammalian forms of the enzyme. The oxidative function of this enzyme allows for the use of chromogens, which when oxidized, not only change color, but precipitate in such a manner as to render a permanent preparation. 

In addition to the many enzyme systems available, there are with each a series of chromogenic substrate solutions that can be used to create different colors and locations of reaction products. For the peroxidase system, there are numerous oxidizable compounds that precipitate as a permanent color. The most common and still widely used is 3,3 diaminobenzidine tetrahydro-chloride (DAB). This compound precipitates to a golden brown color when in solution with peroxidase and hydrogen peroxide. This brown color has many subtleties and readily stands out in a tissue section. With practice, it is possible to differentiate specific from nonspecific staining patterns just by examining the characteristics of the precipitated pigment. This material is also insoluble in alcohol and xylene, and therefore the tissue may be routinely dehydrated and cleared without loss of chromogen. 

There are other chromogens that can be employed when using peroxidase enzymes. The compound 4-chloro-l-naphthol is one that is often used in... 

Bos, E., van der Doelan, A., van Rooy, N., and Schuurs, A. (1981) 3,3, 5,5 -tetra-methylbenzidine as an Ames test negative chromogen for horse-radish peroxidase in enzyme-immunoassay. J. Immunoassay 2, 187-204. 

Variations on the ABC technique can also be used to incorporate different enzymes that result in different chromogenic products. Alkaline phosphatase ABC is one example. The difficulty with this system is the consumption of endogenous alkaline phosphatase, which is more prevalent than peroxidase and is harder to remove. However, the alkaline phosphatase enzyme does provide more product per unit than does peroxidase and is therefore a slightly more sensitive means of detection. Endogenous alkaline phosphatase can be blocked by an incubation in 3 mMlevamisole for 15 min, but some enzyme may escape consumption. Alkaline phosphatase has many substrates, too, the most popular being BCIP/NBT, which precipitates to a dark blue (see Chapter 25). 

A test was conducted to determine if the coverplate plastic had any deleterious effect on the AEG chromogen. Coverplates were finely ground, and the resulting plastic powder was added to AEG. Various concentrations of plastic were used. After 10 min of incubation with the plastic, streptavidin-peroxidase complex was added. After another 5 min, the tubes were photographed. Over a range of added plastic from 0.01 to 0.04 g, there was no detectable decrease in the chromogenic reaction as compared to a tube with no added plastic. 

Uric acid (29) kits Commercial kits are based on the enzymatic process shown in equation 15, followed by a chromogenic oxidation process catalyzed by peroxidase, similar to equation 27, involving 4-aminoantipyrin (81) and 3,5-dichloro-2-hydroxyben-zenesulfonate (88), measuring at 500 to 520 nm . ... 

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