Enzymes chromogenic detection

Make it visible the fluorophore label can be visualized directly using fluorescent microscopy. The biotin label (see Sect. 6.2.1) can be detected using streptavidin conjugated with an enzyme the latter must be visualized through an enzyme chromogenic system. Incubate sections with an appropriate enzyme substrate until optimal color develops (see Sect. 2.3). 

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... 

The Ventana 320 is designed to be used only with detection reagents, enzymes, chromogens, and counterstains supplied by Ventana, but it allows the use of primary antisera not marketed by the company. To use such antisera, however, the company requires purchase of Ventana pipeters and bar codes for each individual antisera. Although this limits a laboratory s ability to be flexible, it assists in assuring quality control of reagents. 

Chromogenic detection of horseradish peroxidase POase is widely used in enzyme immunoassays (EIA) and many suitable chromogens (which are oxidized by the enzyme in the presence of the peroxide or urea peroxide substrates) have been developed. Peroxide is the usual substrate, particularly on solid phases since its reduction results in the formation of inert water near the solid phase (Fig. 7.9). It should be realized that POase has a very pronounced optimum concentration of H2O2 substrate (Tijssen et al., 1982). Activity is low at low substrate concentrations, but inhibition is considerable at high substrate concentrations. The universally used POase, C isozyme, has an optimum in solution of 0.003% peroxide but higher concentrations are usually required on a solid phase. 

A diagnostic method using fluorescence labeled DNA probes to detect and quantify the number complementary chromosomal sequences on a cellular resolution. A related technique that also allows assessment of gene amplifications, but without precise quantification of copy numbers is the chromogenic in situ hybridization (CISH). Here, instead of a fluorescent dye an enzyme that can generate a colored precipitate in the tissue samples is coupled to the DNA probe. 

Recently, two fiuorogenic assays were also described that can be used for detecting BVMO activityHowever, these methods are biased toward specific chromogenic and fiuorogenic substrates and will only yield enzymes that are active toward these and similar compounds. 

Enzymes useful for detection purposes in ELISA techniques (Chapter 26) also can be employed in the creation of highly sensitive DNA probes for hybridization assays. The attached enzyme molecule provides detectability for the oligonucleotide through turnover of substrates that can produce chromogenic or fluorescent products. Enzyme-based hybridization assays are perhaps the most common method of nonradioactive detection used in nucleic acid chemistry today. The sensitivity of enzyme-labeled probes can approach or equal that of radiolabeled nucleic acids, thus eliminating the need for radioactivity in most assay systems. 

The complexities of protocols for fluorescent and chromogenic in situ hybridization necessarily entail careful attention to controls. In particular, the possibility of native enzyme activity or the presence of endogenous biotin in the experimental tissue should be considered, though this can be addressed by exposing control tissue to the detection system in the absence of probe. The relative merits of digoxigenin versus biotin, and some of the technical problems associated with each, have been previously discussed (Chevalier et al., 1997 Luo and jackson, 1999). 

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). 

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