Enzyme assay direct binding

There exists a wide variety in the setup of ELISA assays (direct binding or competition setups) and the enzymatic reaction utilized [148]. A similar principle to enhance sensitivity by enzymatic coupling is realized after gel electrophoretic separation of proteins. Here proteins are transferred to nitrocellulose ( western blot ) and detected by antibody-coupled enzymes. 

Among the biological assays, preference is given to assays that measure a direct interaction between a compound and a target, making data interpretation simpler. Therefore, radioligand binding assays and isolated enzyme assays are preferred to functional or cell-based assays. 

For ELISA, an enzyme is linked chemically to the antibody. The labeled antibody is allowed to bind to the unlabeled antigen, under conditions where nonspecific adsorption is blocked, and any unbound antibody and other proteins are washed away. Binding is detected by a reaction that converts a colorless substrate into a colored reaction product. The color change can be read directly in the reaction tray, making data collection very easy, and ELISA also avoids the hazards of radioactivity. This makes ELISA the preferred method for most direct-binding assays (Plested et al. 2003). 

Among the many immunological assay methods, the enzyme-linked immunosorbent assay methods (ELISA) are the most popular methods. ELISA can detect both antigen molecules and antibody molecules with only a slight modification of the procedure. The direct-binding and sandwich methods that are used for the... 

The determination of the quantity of protein bound to the insoluble carrier sometimes causes difficulties. The methods usually applied are laborious or somewhat inaccurate. Labeling of assayed protein, for instance with C-acet-anhydride, makes it possible to carry out a very fast and exact determination of immobilized protein The determination of bound enzyme C-labeled aldolase after its immobilization on polyacrylamide can serve as an example The concentration measurements of certain proteins are based on their ability to bind certain ligands. Radiolabels such as or H-biotin have been used for the determination of avidin by direct binding or for biotin assay by isotopic dilution Cofactor and fluorescent labeled ligands have been also used for the monitoring of specific protein binding reactions. 

As the result of high specificity and sensitivity, nucleic acid probes are in direct competition with immunoassay for the analytes of some types of clinical analytes, such as infectious disease testing. Assays are being developed, however, that combine both probe and immunoassay technology. In such hybrid probe—immunoassays, the immunoassay portion detects and amplifies the specific binding of the probe to a nucleic acid. Either the probe per se or probe labeled with a specific compound is detected by the antibody, which in turn is labeled with an enzyme or fluorophore that serves as the basis for detection. 

Another commonly used ELISA format is the immobilized antibody assay or direct competitive assay (Eigure 3). The primary anti-analyte antibody is immobilized on the solid phase and the analyte competes with a known amount of enzyme-labeled hapten for binding sites on the immobilized antibody. Eirst, the anti-analyte antibody is adsorbed on the microtiter plate wells. In the competition step, the analyte and enzyme-labeled hapten are added to microtiter plate wells and unbound materials are subsequently washed out. The enzyme substrate is then added for color production. Similarly to indirect competitive immunoassay, absorption is inversely proportional to the concentration of analyte. The direct competitive ELISA format is commonly used in commercial immunoassay test kits. 

Direct and indirect competition formats, illustrated in Figure 1, are widely used for both qualitative and quantitative immunoassays. Direct competition immunoassays employ wells, tubes, beads, or membranes (supports) on to which antibodies have been coated and in which proteins such as bovine semm albumin, fish gelatin, or powdered milk have blocked nonspecific binding sites. Solutions containing analyte (test solution) and an analyte-enzyme conjugate are added, and the analyte and antibody are allowed to compete for the antibody binding sites. The system is washed, and enzyme substrates that are converted to a chromophore or fluorophore by the enzyme-tracer complex are added. Subsequent color or fluorescence development is inversely proportionate to the analyte concentration in the test solution. For this assay format, the proper orientation of the coated antibody is important, and anti-host IgG or protein A or protein G has been utilized to orient the antibody. Immunoassays developed for commercial purposes generally employ direct competition formats because of their simplicity and short assay times. The price for simplicity and short assay time is more complex development needed for a satisfactory incorporation of the label into the antibody or analyte without loss of sensitivity. 

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