Enzyme label peroxidase

Fig. 11. Detection of an antigen-antibody complex using a enzyme label (peroxidase) attached on an immunological basis (according to Stembo-ger...

The primary use of EIA when it was first developed was for histological labeling and localization of specific cell macromolecules. Eor example, enzymes labeled with peroxidase were used to locate specific cellular compartments and stmctures for microscopic examination. The flexibiUty of EIA was recognized quickly and it was adapted for use as a laboratory assay. 

In the most common method for chemiluminescent immunoassay (GLIA), after the immunological reaction and any necessary separation steps, the labeled compounds or complexes react with an oxidizer, eg, hydrogen peroxide, and an enzyme, eg, peroxidase, or a chelating agent such as hemin or metal... 

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). 

Conventional ion-selective electrodes have been used as detectors for immunoassays. Antibody binding measurements can be made with hapten-selective electrodes such as the trimethylphenylammonium ion electrode Enzyme immunoassays in which the enzyme label catalyzes the production of a product that is detected by an ion-selective or gas-sensing electrode take advantage of the amplification effect of enzyme catalysis in order to reach lower detection limits. Systems for hepatitis B surface antigen and estradiol use horseradish peroxidase as the enzyme label and... 

Secondary antibody and determination. A secondary antibody labeled with an enzyme is added which binds to the primary antibody that is bound to the coating antigen. If the primary antibody were produced in a rabbit, an appropriate secondary antibody would be goat anti-rabbit immunoglobulin G (IgG) conjugated with horseradish peroxidase (HRP) (or another enzyme label). Excess secondary antibody is washed away. An appropriate substrate solution is added that will produce a colored or fluorescent product after enzymatic conversion. The amount of enzyme product formed is directly proportional to the amount of first antibody bound to the coating antigen on the plate and is inversely proportional to the amount of analyte in the standards. 

Enzyme labels are usually coupled to secondary antibodies or to (strept)avidin. The latter is used for detection of biotinylated primary or secondary antibodies in ABC methods (see Sect. 6.2.1). Enzyme labels routinely used in immunohisto-chemistry are horseradish peroxidase (HRP) and calf intestinal alkaline phosphatase (AP). Glucose oxidase from Aspergillus niger and E. coli (3-galactosidase are only rarely applied. 

Notes. When using biotin-labeled secondary antibodies instead of enzyme-labeled antibodies, you have first to detect biotin with enzyme-labeled (strept) avidin and proceed further with the Substrate Step (9). Do not add normal serum, non-fat dried milk, culture media or other potential sources of biotin to (strept)avidin-containing reagents. This may result in reduced sensitivity. Solutions containing sodium azide or other inhibitors of peroxidase activity should not be used in diluting the peroxidase substrate. 

If antibodies conjugated to fluorochromes are not desirable, enzyme-labeled antibodies can also be used. In this case, in the presence of a substrate and a chromagen, an enzyme provides the indicator system necessary to visuahze the location of the antibody (Carson, 1997). Horseradish peroxidase (HRP) and alkaline phosphatase (AP) are the enzymes most commonly conjugated to antibodies not labeled by... 

Fig. 2. Diagram illustrating the molecular interactions of the LAB procedure. Horseradish peroxidase is covalently linked to avidin. The primary antibody against the antigen of interest is linked to the enzyme labeled avidin complex (LAB) via a biotinylated secondary antibody raised against immunoglobulin of the animal species used to generate the primary antibody (A, immunoglobulin CCC, long carbon arm extension , biotin A, avidin , peroxidase).

The technique described here is for use with monoclonal primary antibodies of mouse origin, but can easily be adapted for use with polyclonal antibodies from other species (i.e., rabbit). This method uses a secondary biotin-labeled antibody and a detection system that employs a biotin-avidin horseradish peroxidase complex linker step, the so-called ABC (avidin-biotin complex) detection system (5) (see Chapter 25). In this detection system, avidin acts as a bridge between the biotinylated secondary antibody and a biotin-labeled peroxidase enzyme. The anchored enzyme, in the presence of H2O2 can then convert the substrate, diaminobenzidine, to a brown or black reaction product that is easily identifiable in the tissue section. 

The two most common enzyme labels for secondary antibodies are alkaline phosphatase (AP) used in conjunction with the substrate p-nitrophenyl phosphate, which results in a yellow reaction product, and horseradish peroxidase (HRP) used in conjunction with the substrate ABTS and H2O2, which results in blue-green reaction product see Chapter 23). 

Detection reagent (substrate) for enzyme-labeled antibody 2,2-azino-di(3-ethyl-benzthiazohne sulfonate-6) (ABTS) 0.3 g/L in 0.15 M sodium citrate with 0.1% H2O2 for the detection of horseradish peroxidase-labeled secondary antibody or p-nitrophenyl phosphate (pNPP) 1 g/L in 1M diethanolamine in water for the detection of an alkaline phosphatase-labeled antibody (Kirkegaard and Perry Laboratories). 

Tyramide signal amplification (TSA PerkinElmer Life Sciences, Boston) and enzyme-labeled fluorescence (ELF Molecular Probes) are related detection technologies. In the tyramide amplification process, a tyramide-biotin complex is produced by the action of horseradish peroxidase. The complex precipitates near the binding site and accumulates. The complex is detected by the use of streptavidin-Cy3/Cy5. 

Transmission electron microscopy of immunogold labelled sections has shown that the extracellular lignin-degrading enzymes lignin-peroxidase and laccase were localized within the cell wall and mucilage of the hyphae of C. versicolor. Laccase was present in the cell wall layer whereas lignin-... 

As indicator enzymes horseradish peroxidase (HRP or HRPO), alkaline phosphatase (AP), or /i-galactosidase, are favored, since they are relatively robust, have a high product-forming rate, are easy to purify, and are cheap. The most used colloids are from gold, silver, and iron, and iodine isotopes are mostly taken as radioactive labels in immunoassays. 

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. 

Peroxidase is inactivated by anions and certain antimicrobial agents, including azide, cyanide, and thiomersal. Antimicrobial agents may be present in concentrated enzyme label solutions and assay buffers at typically active concentrations, but must not be present in wash solutions or substrate. The latter reagents must be freshly made each day from concentrated stocks. 

The most common enzyme label in IAs and ILAs is horseradish peroxidase (HRP) due to its high turnover number, the sensitivity of its colorimetric and luminometric assays, its suitability for diverse conjugation procedures, and relative small molecular size (40 kDa compared to 100 kDa for alkaline phosphatase). The use of labeled enzymes as tracers allows qualitative and quantitative assay procedures that are not dependent on instrumentation. Thus absorbance, luminescent, electrochemical, or multistage assay systems could be performed (Fig. 10) [23]. 

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],... 

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