Enzyme-linked immunosorbent assay principle

New detection methods of phenolic compounds are being developed. Based on the principle of the enzyme-linked immunosorbent assay (ELISA), a method has been developed to quantify phenolic compounds such as isoflavones (Vergne and others 2007). 

Figure 19-13 illustrates the principle of an enzyme-linked immunosorbent assay, abbreviated ELISA in biochemical literature. Antibody 1, which is specific for the analyte of interest (the antigen), is bound to a polymeric support. In steps 1 and 2, analyte is incubated with the polymer-bound antibody to form a complex. The fraction of antibody sites that bind analyte is proportional to the concentration of analyte in the unknown. The surface is then... 

Particle Concentration Fluorescence Immunoassay. The PCFIA is a solid-phase immunoassay in which proteins are attached to polystyrene particles by adsorption or covalent coupling for the solid phase and fluorescent-labeled reagents are utilized for product detection.22 The general principles of the assay are similar to those of the enzyme-linked immunosorbent assay (ELISA), which has been reviewed extensively elsewhere.23 PCFIAs are performed in specially designed 96-well format Fluoricon assay plates utilizing an automated Screen Machine (Idexx Corporation, Research Product Division, Portland, ME). 

To be able to convey knowledge of the principles and applications of enzyme-linked immunosorbent assays (ELISA). 

The heterogeneous enzyme immunoassays, which include the enzyme-linked immunosorbent assay (ELISA), are based on the same principles as are used in radioimmunoassays (RIA). In short, after incubation of antigen and antibodies, the antigen-antibody complexes formed are separated from free antigen and antibody by one of a number of different techniques, and the activity in one or both of the fractions is determined. 

Alternatively, MIPs have also been used in biological receptors for competitive binding assays. The assay principle is similar to that in other known biological assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA) except that instead of antibodies, MIPs are utilized. This method is often called molecularly imprinted assay (MIA). Typically, in MIA methods, a marker molecule (a labeled analyte analogue) is incubated together with the sample and the MIPs. Analyte and marker molecules compete for the binding... 

On the basis of an enzyme thermistor, Mattiasson et al. (1977) developed one of the first immunosensors. Immobilized antibodies against albumin are placed in a column and set into an ET. After injection of an albumin-sample and a known amount of enzyme-labeled albumin, both are separated from the sample matrix by antibody-antigen-interaction. After injection of a substrate, the change in heat is a measure of analyte concentration. The less heat produced means that more albumin has been bound. An elution step regenerates the ELISA. Due to its thermal detection principle, the procedure is called TELISA (thermometric enzyme-linked immunosorbent assay). Figure 3 shows the principle of the TELISA procedure in its sandwich configuration. 

Test strips, which are available for the determination of about ten low-molecular mass substances (metabolites, drugs, and electrolytes) and eight enzymes [356], can be considered as precursors of optoelectronic biosensors. Efficient optoelectronic sensors based on immobilized dyes have been devised for the determination of glucose, urea, penicillin, and human serum albumin [357]. Other approaches use immobilized luciferase or horseradish peroxidase to assay ATP or NADH or, when coupled with oxidases, to measure uric acid or cholesterol. These principles have not yet been generally accepted for use in routine analysis. Thermistor devices involving immobilized enzymes or antibodies for a number of clinically relevant substances have also been described. Thermometric enzyme linked immunosorbent assays are being routinely employed for monitoring the production of monoclonal antibodies. 

The quantitative aspects of changes in various intracellular pools of NADPH protochlorophyllide oxidoreductase (POR) are important for understanding the principles of operation of the multienzyme system of chlorophyll biosynthesis which proteins are encoded by nuclear DBA and synthesized in the cytoplasm. The application of immunochemical methods /I,2/ allowed to detect loss of POR in the system of intraplastid membranes of etiolated leaves under illumination. In this work the enzyme-linked immunosorbent assay (ELISA) was employed for quantification of POR in different intracellular compartments of postetio-lated and green barley seedlings. 

ELISA or enzyme-linked immunosorbent assays originated from the use of enzyme-antibody (eg. horse-radish peroxidase) complexes for immunohistochemistry. The same principles of spectrophotometric measurement have been employed for the measurement of antibodies (eg. Engvall, 1976 Leinikki and Passila, 1975) or the detection of viruses (Voller et al., 1976b). 

All immunoassays are based on the principle detection method employed. Commonly used meth-of competitive displacement of a labeled drug ods include fluorescence polarization, enzyme im-from an antigen-antibody complex by unlabeled munoassay, cloned enzyme donor immunoassay, drug in the sample. The fundamental difference enzyme-linked immunosorbent assay, and radioin the currently available immunoassays is the immunoassay. 

Figure 14.9 Principle of Enzyme Linked Immunosorbent Assay.

For diagnostic detection of BChE adducts of common G- and V-type nerve agents, enhanced sample throughput was achieved by automated processes in 96-well plate format, extracting plasma by inunimomagnetic separation (Knaack et al., 2012). An alternative method, based on the principle of a sandwich enzyme-linked immunosorbent assay (ELISA), was presented by Wang et al. (2011) to determine OP adducts in complete BChE. 

In addition to these methods, new techniques based on the principle of enzyme-linked immunosorbent assay (ELISA) have recently been introduced for sialidase assay. A glycoprotein or ganglioside substrate is coated in plastic microtiter wells and incubated with an enzyme preparation, followed by detection of the desialylated product using an appropriate ligand. Thus, the activity of Clos-... 

The immunometric-type assay has also been adapted for use with nonisotopic labels and is typically carried out in a heterogeneous format in which the antibody is immobilized on a solid support, such as a microtiter dish, membrane, or collection of beads. The canonical clinical immunoassay format in toady s laboratories is the enzyme-linked immunosorbent sandwich assay, which employs two antibodies, one to capture the analyte and the other to detect and quantify it. More details of the principles of these and other immunoassay techniques are given elsewhere in this encyclopedia. 

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