Enzyme direct-binding method

Direct binding method — If an enzyme is expensive then it is important to use only the amount necessary for detection. Immobilization via the direct binding method (Figure 3.2) involves placing as little as 10 p.1 of enzymatic solution onto the tip of an electrode (a) and then an analogous quantity of the cross-linking... 

Figure 3.2 Immobilization of enzymes by the direct binding method.

As shown in Figure 8.1a, the carrier binding method is based on binding microbial cells directly to water-insoluble carriers. The binding is due to ionic forces between the microbial cells and the water-insoluble carriers. This technique has rarely been used, however, because of lyses dming the enzyme reactions. Microbial cells may leak from the earner, thereby disrupting the immobilisation. Therefore, this method has not been applied successfully.6... 

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. 

There are several modifications to the procedure that allow the target to be identified and quantified. Since the topic is immunocytochemistry, the procedures described deal with ELISAs performed on intact cells fixed onto the wells of 96-well plates. There are two forms of the procedure direct and indirect. The direct method calls for linking the enzyme directly to the antibody of interest. Depending on the availability of the antibody and its activity, direct conjugation of the enzyme to the antibody may interfere with specificity or success of binding with the target. The preferred method is the indirect ELISA. The antigen or cell of interest is immobilized onto the well. The primary antibody (often a... 

The strong alteration of the microenvironment due to covalent fixation is reflected by a change in the kinetic properties of GOD. With different binding methods the pH optimum may be shifted by up to one pH unit in the acid or alkaline direction as compared with the free enzyme. The apparent Am value for glucose may vary in the range 3.1-19.1 mmol/l. 

In case an enzyme does not tolerate direct binding, it may be physically encaged in a macroscopic matrix. To ensure catalytic activity, it is necessary that substrate and product molecules can freely pass into and out of the macroscopic stracture. Due to the lack of covalent binding, entrapment is a mild immobiUzatimi method which is also applicable to the immobilization of viable cells. 

The direct ELISA method is considered the simplest type of immunoassay (Fig. 20). The antigen is adsorbed onto the electrode surface, then an excess of another protein (normally bovine serum albumin, BSA) is added to block all the other binding sites on the surface. An enzyme linked to an antibody in a separate reaction is added, which then produces the enzyme-antibody eomplex. After that, any excess enzyme antibody complex is washed off. The en me substrate is then added and produces an analytical signal directly proportional to the antigen con-eentration in the sample. 

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