Alkaline phosphatase systems

Table 3. Common endogenous enzyme blocking reagents for horseradish peroxidase and alkaline phosphatase systems.

Another common enzyme used m these procedures is alkaline phosphatase. Alkaline phosphatase will cleave phosphates off of a donor molecule, which then in turn acts as a mediator of a color change involving a third molecule. This system is often used because alkaline phosphatase can create more of the color-producing molecules per enzyme molecule than can peroxidase, resulting in better sensitivity. Alkaline phosphatase systems are especially sensitive for examining protein or nucleic acid blots with enzyme labels. The problem when examining tissue is the presence of the endogenous enzyme m the tissues examined. 

Metabolic Functions. Zinc is essential for the function of many enzymes, either in the active site, ie, as a nondialyzable component, of numerous metahoenzymes or as a dialyzable activator in various other enzyme systems (91,92). WeU-characterized zinc metahoenzymes are the carboxypeptidases A and B, thermolysin, neutral protease, leucine amino peptidase, carbonic anhydrase, alkaline phosphatase, aldolase (yeast), alcohol... 

Biomedical Applications. TRIS AMINO is used for a number of purposes in its pure form, it is an acidimetric standard the USP grade can be utilized intraveneously for therapeutic control of blood acidosis TRIS AMINO also is useful in genetic engineering as a buffering agent for enzyme systems, industrial protein purification, and electrophoresis. AMP has found use as a reagent in enzyme-linked immunoassays. The primary appHcation is for alkaline phosphatase assays. 

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. 

Respiratory distress was noted in 2 workers exposed to greater than 40 ppm hydrogen sulfide for under 25 minutes (Spolyar 1951). In animals, impacts on the respiratory system such as increases in the cellularity and lactate dehydrogenase and alkaline phosphatase activities of bronchial lavage fluids have been seen at exposures as low as 10 ppm for 4 hours (Lopez et al. 1987) although without a dose-related trend. 

The major enzymes used in ELISA technology include horseradish peroxidase (HRP), alkaline phosphatase (AP), (3-galactosidase (P-gal), and glucose oxidase (GO). See Chapter 26 for a detailed description of enzyme properties and activities. HRP is by far the most popular enzyme used in antibody-enzyme conjugates. One survey of enzyme use stated that HRP is incorporated in about 80 percent of all antibody conjugates, most of them utilized in diagnostic assay systems. 

Perhaps the most common conjugates of (strept)avidin involve attaching enzyme molecules for use in ELISA systems. As in the case of antibody-enzyme conjugation schemes (Chapter 20), by far the most commonly used enzymes for this purpose are HRP and alkaline phosphatase. Other enzymes such as (3-galactosidase and glucose oxidase are used less often, especially with regard to assay tests for clinically important analytes (Chapter 26). 

Figure 13.8 The etoposide-alkaline phosphatase ADEPT system. Refer to text for specific details...

The RPIA technology has been enhanced in the Stratus CS system by utilization of a dendrimer-antibody complex in which the analyte-specific capture antibody is covalenty coupled onto a dendrimer. The test packs in the Stratus CS system include dendrimer-capture antibody complex reagent, the alkaline phosphatase labeled antibody conjugate reagent, the substrate-wash reagent and a piece of glass fiber filter paper as the solid phase. Preparation and unique properties associated with these dendrimer-coupled antibody complexes are described below. 

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