Detection system phosphatase

The ABC detection system has been shown to be more sensitive than most other detection system (5,6), primarily because of the large size of the preformed ABC complexes, which result in amplification of the signals. Alternative detection systems for immunohistochemical analysis include the peroxidase-antiperoxidase (PAP) (1) and the alkaline phosphatase-antialkaline phosphatase (APAAP) systems (7) (see Chapter 24). These approaches are conceptually and technically similar, and will not be discussed here. 

Detection of the sites of hybridization-dependent binding of biotinylated probe to the filters is most readily conducted with commercially available kits. Favorable results have been obtained with the BluGene Nonradioactive Nucleic Acid Detection System from BRL. Follow the manufacturer s instructions when carrying out the following steps. After washing, sequentially expose the filters to streptavidin and biotinylated alkaline phosphatase (or to a conjugate of these two proteins) This causes the immobilization of alkaline phosphatase at sites of positive hybridization... 

Immunolocalization is a technique for identifying the presence of a protein within the cell, its relative abundance and its subcellular localization. After suitable preparation of the cells, they are treated with an antibody (the primary antibody) that binds to the protein of interest. An antibody that binds to the primary antibody (the secondary antibody) is then allowed to bind and form an antigen—primary antibody—secondary antibody complex. The detection system generally consists of the formation of a colored insoluble product of an enzymatic reaction, the enzyme, such as alkaline phosphatase or horseradish peroxidase, being covalently linked to the secondary antibody. 

Acceptable bridging molecule systems have been developed which have also simplified the utilization of different detection systems. To illustrate this point, a researcher who has developed a unique monoclonal antibody (a primary antibody) in the mouse may select from a variety of commercially available products consisting of different detection systems (e.g. fluorescein, alkaline phosphatase, colloidal gold) attached to an immunoglobulin that will specifically bind to mouse antibodies (a secondary antibody). In this way the researcher may readily obtain and test a number of detection methods for visualizing target-probe interactions without having to directly label the monoclonal antibody probe. For nucleic acid probes, which in themselves are not readily immunodetectable, it is useful to incorporate or attach detectable moieties to the nucleotides. 

Biotin has served this purpose well in both nucleic acid and antibody probe systems. As well as being easily detected with immunoglobulins specific for biotin, biotin may also be detected non-immunologically with avidin or streptavidin, two proteins which share a marked, highly specific affinity for biotin. The affinity constant for avidin-biotin interactions is approximately 10 - liters/mole, much higher than the range for antigen-antibody interactions which are commonly between 10 -10 liters/mole. Consequently, a vast number of detection complexes composed of avidin or streptavidin bound to a detection system are commercially available (e.g. streptavidin-alkaline phosphatase). 

While enzymes may be covalently attached directly to primary probe molecules, as noted above for reasons of reagent versatility, steric factors, and potential signal amplification, indirect detection systems appear to be the more popular. Consequently, enzyme-probe conjugates are typically complexes of a desired enzyme marker and a secondary level probe that is, a probe molecule that can specifically identify a primary level probe molecule, such as an alkaline phosphatase-streptavidin conjugate can identify a biotinylated nucleic acid probe by virtue of the binding affinity between streptavidin and biotin. Other examples of enzyme-probe systems are given in the preceding section on direct and indirect detection systems. 

A low detection limit directly influences the sensitivity of the enzyme-based assay. The final enzyme-substrate interaction must yield an ample amount of some end product which can be accurately monitored and, hopefully, quantitated. The authors experiences have been chiefly with enzymatic detection systems which culminate in a visible chromogenic reaction (e.g. alkaline phosphatase, nitroblue tetrazolium, 5-bromo-4-chloro-3-indolyl phosphate). 

Preparations are incubated with appropriate reagents to allow visualization based upon the detection system associated with the secondary antibody. The secondary antibody may be conjugated to a enzyme (e.g., alkaline phosphatase, horseradish peroxidase). Incubation with the appropriate substrate to the enzyme will result in the production of an insoluble colored product that can be detected upon microscopic analyses of the cells. Secondary antibodies can also be conjugated to fluorochromes (e.g., fluorescein, rhodamine) that can be detected using a microscope equipped to detect fluorescence. Immunohisto-chemistry has proven to be a powerful tool in biochemical toxicology allowing for in situ assessments of protein responses to toxicant exposure. 

Parker, Pardue, and Willis S described a reaction-rate instrument including a reciprocal-time digital computer circuit designed for on-line variable-time analyses. Alkaline phosphatase could be determined satisfactorily with a relative standard deviation of about 1% if the times were in excess of 0.5 s. Their data indicate that, with proper calibration and temperature control, the limitations lie in the chemical and detection systems rather than in the rate-measuring system. 

Some properties of phosphopeptides make them preferable to the native phosphoprotein substrates for use with phosphate detection systems. The values of for peptide substrates are two to three orders of magnitude larger than for protein substrates and allow setting assays with an appropriate substrate concentration using standard phosphate detection approaches. In addition, short synthetic peptides are inexpensive and easy to obtain. Nonetheless, although phosphopeptide substrates are clearly useful in exploring interactions in the immediate vicinity of the phosphatase active site, they are unable to probe distant (allosteric) sites. 

Double immunoenzymatic techniques (Fig. 1.20) permit the demonstration of two antigens concurrently within a single section. As described earlier with reference to alkaline phosphatase methods, double stains were usually performed sequentially however, new polymer-based methods and polyvalent detection systems have made concurrent staining possible. 

Instead of using fluorophore-based detection systems, one can apply the very sensitive detection systems relying on the enz)nne marker horseradish peroxidase (52). Also, other enzyme markers, alkaline phosphatase, and glucose oxidase, and particle-based markers, such as ferritin and colloidal gold, are alternatives and provide complementary detection systems useful for detection of multiple antigens. Here we shall deal only with those techniques that employ peroxidase markers. 

Several variations can be made in the detection system, e.g., using alkaline phosphatase as a reporter enzyme coupled to the tertiary antibody. A two-step reaction, in which the reporter enzyme is coupled to the secondary antibody, may also be used. 

As is illustrated in Sections A and B, HRP is a convenient detection system. Many lectins can be purchased directly conjugated to this and other enzymes (e.g., alkaline phosphatase), enabling a single step overlay. Alternatively, iodinated lectins are commonly used (see Fig. 2). In addition, a wide variety of biotin-lectin conjugates are also available and would be used much in the same manner as is described in Section A, i.e., a two-step procedure (see also Section D). 

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