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Enzymatic detection systems

Applications of enzymatic detection systems in the context of diagnostics are presented in this article. [Pg.226]

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). [Pg.231]

A specific approach for the measurement of base damage to DNA involves the hydrolysis of DNA into monomeric units. Acidic hydrolysis leads to the release of bases while enzymatic treatment yields nucleosides. The resulting mixture of lesions together with the overwhelming presence of normal bases or nucleosides is resolved by chromatography. The targeted damage is then quantified by use of specific detection systems. [Pg.27]

As the enzyme itself is usually the focus of interest, information on the behavior of that enzyme can be obtained by incubating the enzyme with a suitable substrate under appropriate conditions. A suitable substrate in this context is one which can be quantified by an available detection system (often absorbance or fluorescence spectroscopy, radiometry or electrochemistry), or one which yields a product that is similarly detectable. In addition, if separation of substrate from product is necessary before quantification (for example, in radioisotopic assays), this should be readily achievable. It is preferable, although not always possible, to measure the appearance of product, rather than the disappearance of substrate, because a zero baseline is theoretically possible in the former case, improving sensitivity and resolution. Even if a product (or substrate) is not directly amenable to an available detection method, it maybe possible to derivatize the product with a chemical species to form a detectable adduct, or to subject a product to a second enzymatic step (known as a coupled assay, discussed further later) to yield a detectable product. But, regardless of whether substrate, product, or an adduct of either is measured, the parameter we are interested in determining is the initial rate of change of concentration, which is determined from the initial slope of a concentration versus time plot. [Pg.98]

All ee screening systems using UV/Vis spectroscopy as a detection system have a number of advantages, including the possibility of visual pre-screening for activity on microtiter plates in some cases. Moreover, if a reliable UV/Vis signal arises as a consequence of an enzymatic reaction, commercially available UV/Vis plate readers can be used to screen thousands of mutant enzymes catalyzing the reactions of interest in the wells of microtiter plates. [Pg.11]

Many detection systems are based on OH-induced hydroxylation of salicylate (see below). Salicylate, however, inhibits some enzymatic reactions that may... [Pg.58]

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. [Pg.20]

Brennan et al. used a method to detect the reaction of acetylcholineesterase with acetylcholine [46]. The method was based on the use of a monolayer, consisting of fatty acids having Ci6 chain lengths, which were covalently attached to quartz wafers and which contained a small amount of nitrobenzoxadiazole dipalmitoyl phosphatidylethanolamine (NBD-PE) (partitioned from water into the membrane). The enzyme substrate reaction produced a decrease in fluorescence intensity from the monolayer, and the detection system was sensitive to the changes in bulk concentration of as small as 0.1 pM, with a limit of detection of 2 pM of acetylcholine. The mechanism of transduction of the enzymatic reaction was investigated using spectrofluorimetric methods and fluorescence microscopy. [Pg.71]

Multi-step technique (3) This is an indirect/direct method combining unlabeled primary antibodies with directly-conjugated antibodies. The method starts with staining the unlabeled antibody/antibodies with the appropriate detection system, but without performing the final enzymatic staining reaction. The tissue is blocked with normal serum from the host of the first primary antibody before the second, directly-labeled primary antibody is added. The staining ends with the two enzymatic reactions being performed sequentially. [Pg.105]

Lue, R. Y, Zhu, Q., Li, D. (2004). Site-specific peptide immobilization strategies for the rapid detection of kinase activity on microarrays. Site-specific immobilization of biotinylated proteins for protein microarray analysis. Enzymatic profiling system in a small-molecule microarray. Intein-mediated biotinylation of proteins and its application in a protein microarray. Developing site-specific immobilization strategies of peptides in a microarray. Methods Mol. Biol. 278, 191-204. [Pg.158]

Requirements with respect to the label used to mark one of the immunoreagents are comparable to those in other postcolumn reaction detection systems [4]. The label should preferably allow sensitive and rapid detection and be nontoxic, stable, and commercially available. So far, mainly fluorescence labels have been employed (e.g., fluorescein), although, in principle, also liposomes, time-resolved fluorescence, and electrochemical or enzymatic labels are feasible. On the other hand, labels providing a slow response, including radioactive isotopes and glow-type chemiluminescence, are less suitable for immunodetection. [Pg.835]


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