Alkaline enzyme labels

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). 

Instead of immobilizing the antibody onto the transducer, it is possible to use a bare (amperometric or potentiometric) electrode for probing enzyme immunoassay reactions (42). In this case, the content of the immunoassay reaction vessel is injected to an appropriate flow system containing an electrochemical detector, or the electrode can be inserted into the reaction vessel. Remarkably low (femtomolar) detection limits have been reported in connection with the use of the alkaline phosphatase label (43,44). This enzyme catalyzes the hydrolysis of phosphate esters to liberate easily oxidizable phenolic products. 

A sandwich electrochemical enzyme immunoassay has been described for IgG Alkaline phosphatase was again used as the enzyme label with the conversion of phenyl phosphate to phenol being determined electrochemically by LCEC. A detection limit of 10 pg/mL was reported. 

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. 

Enzyme labels are currently by far the most common labels in use. Enzymes include HRP, alkaline phosphatase (AP) and, less often, beta-galactosidase (P-gal). The effectiveness of the conjugated antibody depends on the antibody, the label, and the procedure chosen to link the two.30... 

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... 

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). 

Secondary antibodies are enzyme-linked antibodies directed against the primary antibody host animal s immunoglobulin (usually an IgG). If the primary antibody is mouse anticytokeratin the secondary antibody would be horseradish peroxidase-labeled or alkaline phosphatase-labeled antimouse IgG. 

Figure 6.24 Enzyme-labeled fluorescence. ELF-97 is a soluble phosphorylated substrate cleaved by alkaline phosphatase into a highly fluorescent, insoluble product. (Molecular Probes, Inc., Eugene, OR.)...

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. 

Instead of using human serum albumin as a carrier protein, other workers (135) utilized ovalbumin for preparing the diazotized clenbuterol antigen in an enzyme immunoassay developed for screening of clenbuterol residues in bovine urine, liver, and eye. Alkaline phosphatase rather than -galactosidase was also used as an enzyme label in the preparation of the enzyme-clenbuterol conjugate. 

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]. 

Most electrochemical immunosensors use antibodies or antigens labelled with an enzyme that generates an electroactive product which can be detected at the electrochemical transducer surface. The combination of high enzyme activity and selectivity with the sensitive methods of electrochemical detection provides a basis for the development of immunosensors. Horse radish peroxidase (HRP) and alkaline phosphatase (AP) are popular enzyme labels and can be used with a variety of substrates. 

Enzyme labelling of the detection antibody fill the reservoirs with 15 pL of solution of streptavidin labelled with alkaline phosphatase. Incubate this solution in the micro-channels using 10 multi-loadings so as to ensure the coupling of the biotin moiety of the detection antibody with the ALP-labelled streptavidin. Remove the solution in excess at the end of the pumping process. 

Alkaline phosphatase (EC 3.1.3.1) from bovine intestinal mucosa has proven its worth as an enzyme label for many years. It is stable, has a moderate size (140 kDa), a high turnover number, and can be assayed using a variety of different substrates. Its activity is easily detected by eye in, for example, immunoblots, and it can be quantified by changes in absorbance, fluorescence, or luminescence for use in enzyme-linked immunosorbent assays. 

A variety of techniques (Fig. 1), which use a variable combination of antibody steps, enzyme labels, and chromogens, are employed in immunohistochemistry. The most common immunohistochemical methods are the PAP (peroxidase-antiperoxidase), ABC (avidin-biotin) peroxidase or alkaline phosphatase, and the APAAP (alkaline phosphatase-anti-alkaline phosophatase) techniques however,... 

Dual endogenous enzyme block Horseradish peroxidase and alkaline phosphatase labels... 

Unquenched endogenous alkaline phosphatase activity may be seen in leucocytes, kidney, liver, bone, ovary bladder, salivary glands, placenta and gastro-intestinal tissue. Add levamisole to the alkaline phosphatase chromogen reagent or use another enzyme label such as horseradish peroxidase. Intestinal alkaline phosphatase is not quenched by the addition of levamisole. Pretreat the tissue with 0.03 N HCI. 115-121... 

Stor e characteristics of the native enzyme are generally good, with activities maintained over years. Alkaline phosphatase conjugates are usually prepared via amino or carboxylic acid side chains and purified by gel filtration chromatography. Conjugates are very stable, but the enzyme is costly due to the limited supply of calf intestine. Alkaline (and also neutral and acid) phosphatase enzymes in biological samples are a potential problem with the use of this enzyme label. Careful washing of solid phases may be required to ensure no interference in assays. 

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