Enzyme-linked interaction assay

Noncompetitive Enzyme-Linked Interaction Assay (ELIA)... 

Consequently, in order to determine whether any of the combination of mutations did in fact include binding-site residues, the binding affinities of the mutated cohesins were also evaluated in a quantitative manner. The results are presented in Figure 1. In competitive enzyme-linked interaction assay, cELIA, the native cohesin was used as a standard to coat microtiter plates. The immobilized cohesin was then allowed to interact with an enzyme-linked dockerin solution together with a competitor cohesin (native or mutated) in the solution phase. The measured enzymatic activity, expressed as the percentage of activity detected in the absence of the soluble competitor, reflects die amount of dockerin bound to the immobilized cohesin standard. The IC50, i.e., the... 

Liposome conjugates may be used in various immunoassay procedures. The lipid vesicle can provide a multivalent surface to accommodate numerous antigen-antibody interactions and thus increase the sensitivity of an assay. At the same time, it can function as a vessel to carry encapsulated detection components needed for the assay system. This type of enzyme-linked immunosorbent assay (ELISA) is called a liposome immunosorbent assay or LISA. One method of using liposomes in an immunoassay is to modify the surface so that it can interact to form biotin-avidin or biotin-streptavidin complexes. The avidin-biotin interaction can be used to increase detectability or sensitivity in immunoassay tests (Chapter 23) (Savage et al., 1992). 

The model immunoassay is the enzyme-linked immunosorbent assay (ELISA) in which a non-specific capture antibody is bound to a surface, such as a multi-well plate or small tube [13]. In the basic form of ELISA, a second antibody tagged with an enzyme interacts specifically with the analyte. The enzyme assay produces a colored product that is read with a spectrophotometer. There are many variations on the basic immunoassay format that serve to increase sensitivity, specificity, linear range, and speed. Many commercial instruments have been developed to take advantage of various technologies for reporter molecules. The immunoassay may be coupled to an electronic sensor and transducer, such as a surface acoustical wave (SAW) sensor. Electrochemiluminescence (ECL) is a method in which the detector antibody is tagged with a ruthenium-containing chelate [13-15]. When the tag is... 

Enzyme-linked immunosorbent assay (ELISA) is a new method in alkaloid studies. The application of ELISA to alkaloid study is based on antibody incubations. It differs from classical precipitation-based methods in that specific antigen-antibody interactions are recognized by assaying an enzyme label conjugated to one reactant, usually an antibody. Because of the sensitivity with which enzyme... 

The most predominant application of antibodies has been in the area of diagnostic assays, known as immunoassays, which exploits the specific interaction between the antibody and antigen. The world immunoassay market for clinical and food diagnostics, environmental analysis and other applications exceeded 1.2 billion in 1990. ELISA (Enzyme-Linked Immunosorbant Assay) represents 60% of that market (2). Annual growth rates have been projected at 10-15%. The food diagnostics is projected to grow to 500 million by the year 2000 (3). 

The traditional methods of evaluating the interactions of glycans and glycan-binding proteins are based on isothermal titration calorimetry, surface plasmon resonance (SPR), and enzyme-linked lectin assays. Recently, glycan arrays have been developed as a... 

Figure 2-13. Principle of radioimmuno assay (RIA) and enzyme-linked immunoabsorbent assay (ELISA). RIA and ELISA both depend on the highly specific interaction of an antibody with an antigen to determine, for example, the amount of antigen immobilised on a surface. In the example illustrated above, that is achieved by first binding a specific antibody to the surface-bound antigen, and then adding a second antibody which binds specifically to the first. For RIA, the second antibody is radioactively...

Fig. 10.10. Determination of thermogenin amount in brown adipose tissue mitochondria by the enzyme-linked immunosorbent assay (ELISA) system. The amount of thermogenin was determined as elsewhere described (Cannon et al. [13] Sundin et al. [40] Hansen et al. [56]) in an assay system based on the competition between absorbed and added thermogenin for rabbit on/r-rat-thermogenin antibodies. The interaction was followed with a sheep onri-rabbit-IgG antibody conjugated to alkaline phosphatase. The reaction was linearized as indicated (abs 0 is the absorbance developed in the absence of competing thermogenin). It is seen that this assay can detect less than 0.25 fig thermogenin, i.e., the content in less than 10 fig of mitochondria. It is also seen that the thermogenin content of rat brown fat mitochondria is approximately doubled after a 24 h cold stress. (Our unpublished observations.)...

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. 

The release of different cytokines and chemokines is often measured in the supernatant of NM-exposed cells in order to estimate the pro-inflammatory potential of NMs. It is usually done using a reliable and very sensitive enzyme-linked immunosorbent assay (ELISA). Since the presence of NMs could interact with the optical readouts of ELISA, the cell supernatants are centrifuged to sediment the NMs. However, due to the high affinity of the different proteins to NM surface, also cytokines can adsorb on the particles and be removed during the centrifugation, so their production can be underestimated [59, 65]. 

Antibodies can be used for a variety of applications in the molecular characterization of receptors and receptor-hgand interactions. Antibodies can be used for the detection of receptors in tissue shces. Western blot experiments [48], or ELISAs (enzyme-linked immunosorbent assays) [49]. They can also be used in competition experiments to map the binding epitope of a hgand [50]. Even though the use of antibodies is routine, fhere is no general protocol for fheir generation. 

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