Assay using immobilized enzymes

Figure 3 Example of a typical luminescent flow sensing device [manifold for pesticide chemiluminescent flow assay with one (A) or two (B) columns using immobilized enzymes]. A = immobilized dehydrogenase (Phe-DH) 1.0 m coil B = immobilized biolumi-...

There are many instruments designed for either enzyme assays or substrate assays using enzymes. Information on the analytical capabilities of these instruments will be supplied by the manufacturers. This will often include protocols for specified assays using kits of commercially available pre-prepared reagents. These may be in liquid or dry form and may, for substrate assays, include immobilized enzymes. The facility to be able to develop additional automated methods on a particular instrument will depend upon its design and some instruments are dedicated solely to specified analyses. 

It has been shown that nanometer-scale PMBN structures can be formed on the surface of an electrode using an electrospray deposition (ESD) method, enlarging the surface area available for conjugation of biomolecules. A much larger quantity of biomolecules could be immobilized on the ESD surface compared with a planar spin-coated surface. This resulted in a significant increase in the signal from an enzyme-linked immunosorbent assay (ELISA) carried out using immobilized enzymes. ... 

A review on the subject of immobilized enzymes in biochemical analysis covers preparation and properties of immobilized enzymes assay methods using immobilized enzymes (spectrophotomatic assays, automated methods in biochemical analysis, enzyme electrodes, and colorimetric analyses using immobilized enzymes) and applications of immobilized enzymes in biochemical analysis. ... 

In fact, most RIAs and many nonisotopic immunoassays use a competitive binding format (see Fig. 2). In this approach, the analyte in the sample to be measured competes with a known amount of added analyte that has been labeled with an indicator that binds to the immobilized antibody. After reaction, the free analyte—analyte-indicator solution is washed away from the soHd phase. The analyte-indicator on the soHd phase or remaining in the wash solution is then used to quantify the amount of analyte present in the sample as measured against a control assay using only an analyte-indicator. This is done by quantifying the analyte-indicator using the method appropriate for the assay, for example, enzyme activity, fluorescence, radioactivity, etc. 

For luciferin, a firefly luciferase cosubstrate, another method of retention has been evaluated which consisted of incorporating the substrate in acrylic microspheres during their formation, these last being then confined in a polymeric matrix31. Using the suitable co-immobilized enzymes (adenylate kinase and creatine kinase), the three adenylic nucleotides (ATP, ADP and AMP) could be assayed continuously and reproducibly with a selfcontainment working time of 3 h. 

Total AH = —208 kJ mol-1 Because the energy changes are so small, microcalorimetry lends itself to substrate assays in the presence of excess enzyme rather than to enzyme assays and is particularly useful when immobilized enzymes are used. 

Immobilized enzymes may be used in affinity chromatographic methods but their use as catalysts may be in either the production or removal of compounds in chemical processes or as analytical tools. Many substrate assays can be performed using enzymes immobilized on a variety of surfaces, e.g. glass beads, plastic or nylon tubing. Alternatively they may be incorporated into gel or microparticulate layers on dry strips or slides. 

In this paper we have immobilized an enzyme within a thermally reversible hydrogel. Immobilized enzymes have been used in a variety of applications, ranging from treatment of diseases to sensors, assays, and industrial processes (15-20). When an enzyme is immobilized within a gel which exhibits reversible shrinking and swelling as the ten rature is raised and lowered through the LCST of the gel matrix polymer, the enzyme may be switched off and on as the substrate diffusion rate is regulated by the gel pore size (5). In adcfition to enzymes, a variety... 

The LDH+ALT reactor provided a linear response from 0.1 to 50 pmol/L lactate, thereby increasing lactate conversion by 117-183% relative to LDH alone. The intra- and inter-assay CV were both less than 5%, and recoveries ranged from 93 to 106%. Even though roughly 100% of the LDH and ALT added bound to the support under the immobilization conditions used, the activities of the immobilized enzymes were ca. 3% of those of the free enzymes, which is consistent with previous results obtained by the same [67] and other authors [69,70]. Jointly immobilized LDH and ALT preserved ca. 50% of their original activity after 60-90 days of intermittent use. On the other hand, immobilized luciferase was less markedly inhibited than that in the free solution by substances present in the biological samples assayed [71]. 

Figure 8.2. Schematic representation of a competitive enzyme-linked immunosorbent assay using (a) immobilized antigen or (b) immobilized antibody.

Before an immobilized enzyme can be used for an industrial process, it is essential to characterize it in terms of its catalytic and kinetic properties. A quantitative assay must be developed to measure the activity, kinetic parameters, and stability of the enzyme. In a coupling reaction, H202 rapidly reacts with phenol and 4-aminoantipyrine (electron donor) in the presence of peroxidase to produce a quinoneimine chromogen (Equation E12.2, Figure El 1.2), which is intensely colored with a maximum absorbance at 510 nm. (This is the same as the product formed in the analysis of cholesterol in Experiment 11.)... 

In addition to assay features already mentioned, other factors may influence the choice of assay by the user. In terms of sensitivity of the assay, the threshold of detection of lipase activity, using the procedures as described in this unit, is on the order of 10 2 U for titrimetry, 10H U for colorimetry, and 10 4 U for spectrophotometry (where U is the amount of enzyme required to yield 1 imol product per minute). The smallest amounts (volumes) of materials, including enzyme, are required for the spectrophotometric method, and progressively more material is required for the colorimetric and titrimetric methods. Unless a flow cell adapter is available, the spectrophotometric method is not suitable for analysis of particulate (immobilized) enzyme preparations, whereas the other assay procedures are. 

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